Nav2 channel gene-deficient non-human animals

ABSTRACT

An object of the present invention is to provide a null mutant non-human animal showing salt intake behavior similar to that of wild-type animals under water-sufficient conditions and showing much more intakes of hypertonic saline compared with wild-type animals under water- and salt-depleted conditions, for example, an Na v 2 gene-deficient non-human animal, which is useful as a model animal of excessive salt intake experiments.  
     The object will be attained by following process: mouse genomic libraries are screened with rat NaG cDNA as a probe, then Na v 2 gene of genomic DNA is isolated, and a targeting vector is constructed by inserting marker gene such as neo gene into the exon of Na v 2. After thus constructed targeting vector is induced to ES cells, homologously recombined ES cells are selected, then germ line chimeric mice are constructed with this ES cells strain, and they are hybridized with the wild-type mice and heterozygous mutant mice are obtained. By intercrossing of thus obtained heterozygous mutant mice, Na v 2 knockout mice are constructed.

TECHNICAL FIELD TO WHICH THE INVENTION PERTAINS

[0001] This invention relates to a non-human animal whose function of Na_(v)2 channel gene is deficient on its chromosome that shows salt intake behavior similar to that of wild-type animals under water-sufficient conditions and shows much more intakes of hypertonic saline compared with wild-type animals under water- and salt-depleted conditions, a protein acting as a sensor of extracellular sodium ion level, a gene that codes for said protein, and the like.

PRIOR ART

[0002] Voltage-dependent sodium channels as well as voltage-dependent potassium channels are known as the ion channels which play the main role in the generation and the propagation of action potential in excitable cells such as nerve cells, muscle cells, and the like. A sodium channel molecule comprises an ion-selective channel with voltage-sensors, and is consisted of α-subunits comprised of glycoprotein of 270 kDa, and 1 or 2 smaller β-subunits. Voltage-dependent sodium channels are closed when the cell membrane is in resting potential (normally −70 to −90 mV), but they open when the cell membrane depolarizes and close approximately 1 msec later. Therefore, it is said that a sodium channel protein molecule has a voltage-sensor that senses the membrane potential, and opens the channel, a selective filter for filtering a sodium ion selectively, and inactivation gate.

[0003] Since the identification of a sodium channel protein α-subunit cDNA type I, II, and III in the brain (Nature 320, 188-192(1986), FEBS Lett. 228, 187-194(1988)) by the inventors of the present invention, multiple structurally related isoforms of the α-subunit have been cloned from various tissues, forming a multigene family. In addition to the excitable cells, it has recently been found that glial cells also express voltage-sensitive sodium currents (Trends Neurosci. 19, 325-332(1996)). In situ hybridization, RT-PCR, Northern blot analysis and immunocytochemistry have clearly demonstrated the presence of brain-type I, II, III, H1, Na_(s), NaCH6 and the like in glial cells (Glia 26, 92-96(1999)). However, the functional roles of these voltage-dependent sodium channels in so-called electrically inexcitable cells have not yet been delineated.

[0004] Several years ago, a partial cDNA homologous to the voltage-dependent sodium channel α-subunit was cloned from a cDNA library derived from rat astrocytes, and designated NaG (Proc. Natl. Acad. Sci. USA 89, 7272-7276(1992)). Subsequently, similar α-subunit isoforms were independently cloned from various animal species: Na_(v)2.1 from human heart (Proc. Natl. Acad. Sci. USA 89, 4893-4897(1992)), Na_(v)2.3 from a mouse arterial tumor cell line (J. Biol. Chem. 269, 30125-30131), and SCL11 from rat dorsal root ganglia which corresponds to splicing variant of NaG (FEBS Lett. 400, 183-187(1997)). From the sequence homology, it is possible to assume that they are species orthologues and to classify them into another subfamily of α-subunit of voltage-dependent sodium channels (NaCh), namely, subfamily 2 NaCh (Na_(v)2). Their overall amino acid sequences had less than 50% identity with those of the previously cloned voltage-dependent sodium channels, and the sequences are characterized as rather unique even in the regions associated with ion selectivity and voltage-dependent activation and inactivation. Such regions are perfectly conserved in all other subfamily members, suggesting that the Na_(v)2 has specific channel properties. However, all the attempts to express functional Na_(v)2 channels in heterologous expression systems using such as Xenopus oocytes, CHO cells, HEK293 cells and the like have been unsuccessful, and the function of Na_(v)2 channels in vivo has been totally unknown.

[0005] NaG/SCL11 was originally thought to be one of the voltage-dependent sodium channels (NaChs) expressed in astrocytes because it was cloned from astrocytes, but subsequent in situ hybridization studies revealed that Na_(v)2 is expressed not in astrocytes but in Schwann cells and the spinal sensory neurons in vivo (Glia 21, 269-276(1997)). Relatively high levels of NaG mRNA are detected outside of the nervous system, particularly in lung and heart. In addition, RNase protection and Northern blot analyses demonstrated the presence of NaG mRNA in the central nervous system. However, it has been reported that NaG mRNA was not detectable by in situ hybridization using a non-isotopic probe except in the mesencephalic nucleus V. (Mol. Brain Res. 45, 71-82(1997)), suggesting that NaG mRNA is broadly expressed at a low level throughout the central nervous system or expressed restrictedly in specific regions in the central nervous system. The distribution of the NaG channel among these diverse tissues and cell-types including electrically inexcitable cells, suggests a role for this channel other than in action-potential generation and propagation.

AN OBJECT TO BE ATTAINED BY THE INVENTION

[0006] So far, a model animal for excessive salt intake experiments showing salt intake behavior similar to that of wild-types under water-sufficient conditions and showing abnormal intakes of hypertonic saline compared with wild-types under water- and salt-depleted conditions has not been known. A protein acting as a sensor of extracellular sodium ion level, and a gene that codes for such protein have not been known as well. The object of the present invention is to provide a null mutant non-human animal showing salt intake behavior similar to that of wild-type animals under water-sufficient conditions and showing much more intakes of hypertonic saline compared with wild-type animals under water- and salt-depleted conditions, for example, an Na_(v)2 gene deficient non-human animal, which is useful as said model animal of excessive salt intake experiments, a protein acting as a sensor of extracellular sodium ion level, and a gene that codes for said protein.

MEANS TO ATTAIN THE OBJECT

[0007] The inventors have been intensely studied the function and the role of Na_(v)2 channel in viva, which was unknown, and generated Na_(v)2 channel knockout mice, then confirmed that Na_(v)2 channel plays a role to sense and control sodium ion level in body fluids. Subsequently, it has been found that these Na_(v)2 channel knockout mice show salt intake behavior similar to that of wild-types under water-sufficient conditions and show abnormal behavior such as much more intakes of hypertonic saline compared with wild-types under water- and salt-depleted conditions, and thus the present invention has been completed.

[0008] In other words, this invention relates to a null mutant non-human animal characterized in showing salt intake behavior similar to that of wild-type animals under water-sufficient conditions and showing much more intakes of hypertonic saline compared with wild-type animals under water- and salt-depleted conditions (claim 1), the null mutant non-human animal according to claim 1, wherein the function of Na_(v)2 gene is deficient on its chromosome (claim 2 ), the null mutant non-human animal according to claim 2, wherein the non-human animal is a rodent (claim 3), and the null mutant non-human animal according to claim 3, wherein the rodent is a mouse (claim 4).

[0009] This invention also relates to a gene that codes for a protein acting as a sensor of extracellular sodium ion level (claim 5), the gene according to claim 5, wherein the protein is comprised of amino acid sequence shown in Seq. ID No. 3, or is comprised of amino acid sequence where one or a few amino acids are deficient, substituted, or added, in amino acid sequence shown in Seq. ID No. 3 (claim 6), the gene according to claim 5, which is comprised of DNA that contains a base sequence shown in Seq. ID No. 2 or its complimentary sequence, and a part or whole of those sequences (claim 7), and the gene according to claim 5, which is comprised of DNA being hybridized under stringent conditions with DNA that contains a base sequence shown in Seq. ID No. 2 or its complimentary sequence, and a part of or whole of those sequences (claim 8).

[0010] This invention relates to a protein acting as a sensor of extracellular sodium ion level (claim 9), the protein according to claim 9, which is comprised of amino acid sequence shown in Seq. ID No. 3 (claim 10), the protein according to claim 9, which is comprised of amino acid sequence where one or a few amino acids are deficient, substituted, or added, in amino acid sequence shown in Seq. ID No. 3 (claim 11), a fusion protein created by combining a protein acting as a sensor of extracellular sodium ion level and a marker protein and/or a peptide tag (claim 12), and the fusion protein according to claim 12, wherein the protein acting as a sensor of extracellular sodium ion level is the protein according to claims 10 or 11 (claim 13).

[0011] This invention also relates to an antibody which specifically combines with a protein acting as a sensor of extracellular sodium ion level (claim 14), the antibody according to claim 14, wherein the protein acting as a sensor of extracellular sodium ion level is the protein according to claims 10 or 11 (claim 15), and the antibody according to claims 14 or 15, wherein the antibody is a monoclonal antibody (claim 16).

[0012] This invention relates to a host cell which contains an expression system that can express a protein acting as a sensor of extracellular sodium ion level (claim 17), and the host cell according to claim 17, wherein the protein acting as a sensor of extracellular sodium ion level is the protein according to claims 10 or 11 (claim 18).

[0013] This invention also relates to a transgenic non-human animal which excessively expresses a protein acting as a sensor of extracellular sodium ion level (claim 19), the transgenic non-human animal according to claim 19, wherein the protein acting as a sensor of extracellular sodium ion level is the protein according to claims 10 or 11 (claim 20), and the transgenic non-human animal according to claims 19 or 20, wherein the non-human animal is a mouse or a rat (claim 21).

[0014] This invention relates to a method of screening a material that promotes or suppresses the function or the expression of a protein acting as a sensor of extracellular sodium ion level characterized in using a cell that expresses a protein acting as a sensor of extracellular sodium ion level, and a subject material (claim 22), the method of screening a material that promotes or suppresses the function or the expression of a protein acting as a sensor of extracellular sodium ion level according to claim 22, wherein the cell that expresses a protein acting as a sensor of extracellular sodium ion level is the host cell according to claims 17 or 18 (claim 23), and a method of screening a material that promotes or suppresses the function or the expression of a protein acting as a sensor of extracellular sodium ion level characterized in using the non-human animal according to any one of claims 1 to 4 or the non-human animal according to any one of claims 19 to 21, and a subject material (claim 24).

[0015] This invention relates to a material that promotes or suppresses the function or the expression of a protein acting as a sensor of extracellular sodium ion level characterized in being available through the screening method according to any one of claims 22 to 24 (claim 25), a medical compound used for curing patients who need promotion of the function or enhancement of the expression of a protein acting as a sensor of extracellular sodium ion level, and containing the protein according to any one of claims 9 to 11 or the material that promotes the function or the expression of a protein acting as a sensor of extracellular sodium ion level according to claim 25 as its effective components (claim 26), and a medical compound used for curing patients who need suppression of the function or the expression of a protein acting as a sensor of extracellular sodium ion level, and containing the protein according to any one of claims 9 to 11 or the material that suppresses the function or the expression of a protein acting as a sensor of extracellular sodium ion level according to claim 25 as its effective components (claim 27).

BRIEF EXPLANATION OF DRAWINGS

[0016] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0017]FIG. 1 is a view showing a gene map of Na_(v)2 knockout mice of the present invention (a), the result of Southern blot technique (b), genomic PCR (c) and Western blot technique (d) of genomic DNA.

[0018]FIG. 2 is a view showing lacZ-expressing areas in embryos, dorsal root ganglia and the thoratic regions of Na_(v)2 knockout mice of the present invention.

[0019]FIG. 3 is a view showing lacZ-expressing areas in the brains of Na_(v)2 knockout mice of the present invention.

[0020]FIG. 4 is a view showing the result of the influence of dehydration on the expression of a nuclear protein, Fos, in Na_(v)2 knockout mice of the present invention.

[0021]FIG. 5 is a view showing the result of the influence of mouse Na_(v)2 channel deficiency of the present invention to water and salt intake of mice.

[0022]FIG. 6 is a view showing the result of responses to various tastant stimuli in the chorda tympani nerve of Na_(v)2 knockout mice of the present invention.

[0023]FIG. 7 is a view showing the result of the measurement of preference ratio for 0.3 M NaCl solution and total fluid intake of Na_(v)2 knockout mice of the present invention before and after 24-h dehydration.

[0024]FIG. 8 is a view showing the result of the sodium-depletion induced salt appetite test for Na_(v)2 knockout mice of the present invention.

MODE FOR CARRYING OUT THE INVENTION

[0025] The null mutant non-human animal of the present invention is not particularly limited, any non-human animal showing salt intake behavior similar to wild-types under water-sufficient conditions and showing much more intakes of hypertonic saline compared with wild-types under water- and salt-depleted conditions will suffice, however, the null mutant non-human animal whose function of Na_(v)2 genes is deficient on its chromosome is exemplified as a concrete example of such non-human animal. “Much more intakes of hypertonic saline compared with wild-type animals under water- and salt-depleted conditions” mentioned here means that, for example, in case of mice, the intake behavior in which the intake amount of 0.3 M saline after 24-h dehydration increases by 1.5-times or more, preferably by 2-times or more compared with that of the wild-type animals, preferably the littermate wild-type animals. Further, “the null mutant non-human animal whose function of Na_(v)2 genes is deficient on its chromosome” means the non-human animal whose endogenous genes that code for Na_(v)2 are inactivated by its destruction, deficiency, substitution or the like, so that the animal has lost its function of expressing Na_(v)2, and a rodent such as a mouse or a rat and the like is exemplified as a concrete example of the non-human animal, however, the non-human animal is not limited to the exemplification. In following explanation, a mouse is cited as an example of the non-human animal.

[0026] Any constructing method of Na_(v)2 knockout mice will suffice as long as it can construct the knockout mice that have lost the function of expressing Na_(v)2. For instance, the following method is exemplified; the genomic DNA library of mice is screened by using cDNA that codes for rat NaG, which is the species counterpart of mice Na_(v)2, as a probe, then an Na_(v)2 gene of genomic DNA is isolated, subsequently a targeting vector is constructed by inserting a marker gene such as neo gene or the like into the exon of Na_(v)2, and thus constructed vector is induced to ES cells by electroporation method, then homologously recombined ES cells are selected, and germ line chimeric mice are constructed with this ES cells strain, and they are intercrossed with the wild-type mice, then the heterozygous mutant mice (F1: first filial generation) are obtained, and by intercrossing of those heterozygous mutant mice, wild-type mice, which are littermates of Na_(v)2 knockout mice generated according to Mendelian ratio, can be generated,.

[0027] The protein acting as a sensor of extracellular sodium ion level of the present invention is not particularly limited as long as it acts as a sensor of sodium ion level in nerve cells of the brain. For example, Na_(v)2 shown in Seq. ID No. 3 (GenBank accession number: L36179) or a protein comprised of amino acid sequence where one or a few amino acids are deficient, substituted, or added in amino acid sequence shown in Seq. ID No. 3 and acts as a sensor of extracellular sodium ion level, or a recombined protein of said proteins are concretely exemplified. Said protein acting as a sensor of extracellular sodium ion level can be prepared based on the DNA sequence information and the like by publicly known methods.

[0028] The genes of the invention that code for a protein acting as a sensor of extracellular sodium ion level include the gene that codes for Na_(v)2 shown in Seq. ID No. 3 in the sequence listing, for instance, Na_(v)2 gene shown in Seq. ID No. 2, gene DNA that codes for a protein comprised of amino acid sequence where one or a few amino acids are deficient, substituted, or added in amino acid sequence shown in Seq. ID No. 3, and the DNA that hybridizes with these gene DNAs under the stringent conditions and codes for the protein acting as a sensor of extracellular sodium ion level. These genes can be prepared based on the DNA sequence information and the like, for example with genomic library of mice constructed from cell line R1, 129/SvJ mice gene library or the like, by publicly known methods.

[0029] It is also possible to obtain DNA of the target that codes for a protein acting as a sensor of extracellular sodium ion level, such as human Na_(v)2.1 (GenBank accession number: M91556), rat NaG/SCL11 (GenBank accession number: Y09164) and the like, which have the same effect as Na_(v)2 genes, by hybridizing DNA library derived from mice under the stringent conditions, with a base sequence shown in Seq. ID No. 2 or its complimentary sequence and a part or whole of those sequences as a probe, and subsequent isolation of DNA that hybridizes said probe. As conditions of the hybridization to obtain said DNA, for instance, a hybridization at 42° C. and a rinsing treatment in buffer including 1×SSC and 0.1% SDS at 42° C., and more preferably, a hybridization at 65° C. and a rinsing treatment in buffer including 1×SSC and 0.1% SDS at 65° C. are exemplified. As factors influential in the stringency of a hybridization, there are various factors besides the above-mentioned temperature condition, and it is possible for a person skilled in the art to realize a same stringency as the above-illustrated stringency of the hybridization by combining those various factors appropriately.

[0030] The fusion protein of the present invention is defined as a protein constructed by combining a protein acting as a sensor of extracellular sodium ion level, such as Na_(v)2 and the like, and a marker protein and/or a peptide tag. As a marker protein, any of conventionally known marker protein will suffice, for example, alkaline phosphatase, Fc region of antibodies, HRP, GFP and the like are concretely exemplified, and as a peptide tag of the present invention, conventionally known peptide tag such as Myc tag, His tag, FLAG tag, GST tag and the like are concretely exemplified. Said fusion proteins can be constructed by a usual method, and are useful as an investigational reagent in the field concerned such as the purification of a protein acting as a sensor of extracellular sodium ion level utilizing the affinity between Ni-NTA and His tag, the detection of said protein, the quantification of an antibody to said protein and the like.

[0031] As an antibody of the present invention that specifically combines with a protein acting as a sensor of extracellular sodium ion level, an immunospecific antibody such as a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a single stranded antibody, a humanized antibody and the like are concretely exemplified. Though these antibodies can be constructed by a usual method with the above-mentioned protein acting as a sensor of extracellular sodium ion level as an antigen, a monoclonal antibody is more preferable among them because of its specificity. Said antibody that specifically combines with a protein acting as a sensor of extracellular sodium ion level, such as a monoclonal antibody or the like, is useful, for instance, for the diagnosis of diseases caused by mutation or deficiency of Na_(v)2 such as chronic diseases of human caused by excessive intake of salt, and for elucidation of molecular mechanism of a protein such as Na_(v)2 or the like acting as a sensor of extracellular sodium ion level.

[0032] An antibody to a protein acting as a sensor of extracellular sodium ion level is developed by administering fragments containing a protein acting as a sensor of extracellular sodium ion level or its epitope, or cells that express said protein on the surface of the membrane to animals (preferably excluding human) with usual protocol. For instance, a polyclonal antibody can be prepared by immunizing a rabbit or the like with an antigen peptide as an immunogen, constructing antiserum by a usual method, and then purifying the constructed antiserum by a column in which the peptide used as the immunogen is fixed. Further, in preparation of a monoclonal antibody, any method that brings antibodies developed by cultured materials of continuous cell line, such as hybridoma method (Nature 256, 495-497, 1975), trioma method, human B-cell hybridoma method (Immunology Today 4, 72, 1983), and EBV-hybridoma method (MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985) can be used. Followings are the explanation of the method of constructing a monoclonal antibody that specifically combines with Na_(v)2 derived from mice, that is, an anti-mNa_(v)2 monoclonal antibody, with an example of Na_(v)2 derived from mice as a protein acting as a sensor of extracellular sodium ion level.

[0033] The above-stated anti-mNa_(v)2 monoclonal antibody can be developed by cultivating an anti-mNa_(v)2 monoclonal antibody developing hybridoma in vivo or in vitro by a usual method. For instance, said anti-mNa_(v)2 monoclonal antibody is available through cultivation inside abdominal cavity of rodents, preferably mice or rats, in vivo, and through cultivation on a culture medium for animal cells in vitro. As examples of a medium for cultivating hybridoma in vitro, some cell culture mediums, such as RPMI1640, MEM or the like, which contain antibiotics like penicillin, streptomycin or the like are exemplified.

[0034] Anti-mNa_(v)2 monoclonal antibodies developing hybridoma can be constructed, for example, by the following procedures. First, BALB/c mice are immunized by using Na_(v)2 derived from mice or the like, then spleen cells and mouse NS-1 cells (ATCC TIB-18) of the immunized mice are fused by a usual method, and then screened by immunofluorescence staining pattern. As methods of separating and purifying said monoclonal antibodies, any method generally used to purify a protein will suffice, and liquid chromatography such as affinity chromatography and the like are concretely exemplified.

[0035] In order to develop a single stranded antibody to a protein acting as a sensor of extracellular sodium ion level of the present invention, the preparation method of single stranded antibodies (U.S. Pat. No. 4,946,778) can be applied. Further, in order to express a humanized antibody, it is possible to use transgenic mice, other mammalian animals or the like, and to isolate and identify the clones that express a protein acting as a sensor of extracellular sodium ion level with the above-mentioned antibodies, and to purify the polypeptide by affinity chromatography. An antibody to a protein acting as a sensor of extracellular sodium ion level is useful for elucidating the molecular mechanism of a protein acting as a sensor of extracellular sodium ion level.

[0036] It is possible to analyze the function of said protein acting as a sensor of extracellular sodium ion level by using the above-mentioned antibodies such as anti-mNa_(v)2 monoclonal antibodies and the like labeled with fluorescent materials like FITC (fluorescein isothiocyanate), tetramethylrhodamine isothiocyanate or the like; with radioisotopes such as ¹²⁵I, ³²p, ¹⁴C, ³⁵S, ³H or the like; or with enzymes like alkaline phosphatase, peroxidase, β-galactosidase, phycoerythrin or the like; or by using fusion proteins where anti-mNa_(v)2 monoclonal antibodies are fused with fluorescence emission proteins such as green fluorescent protein (GFP) or the like. Examples of the immunoassays include RIA method, ELISA method, fluorescent antibody technique, plaque method, spot method, hemagglutination reaction method, Ouchterony method and the like.

[0037] This invention relates to a host cell which contains an expression system that can express a protein acting as a sensor of extracellular sodium ion level. The gene that codes for a protein acting as a sensor of extracellular sodium ion level can be introduced into a host cell by a number of methods described in standard laboratory manuals by Davis et al. (BASIC METHODS in MOLECULAR BIOLOGY, 1986), and by Sambrook et al. (MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and the like. Examples of those methods include calcium phosphate transfection, DEAE-dextran-mediated transfection, transvection, microinjection, cationic liposome-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection. Examples of the host cells include bacterial procaryotic cells such as Escherichia coli, Streptomyces, Bacillus subtilis, Streptococcus, Staphylococcus and the like; fungous cells such as yeast, Aspergillus and the like; insect cells such as drosophila S2, spodptera Sf9 and the like; and animal or plant cells such as L cells, CHO cells, COS cells, HeLa cells, C127 cells, BALB/c3T3 cells (including mutant strains deficient in dihydrofolate reductase, thymidine kinase or the like), BHK21 cells, HEK293 cells, Bowes melanoma cells, oocytes and the like.

[0038] As the expression system, any expression system that can express a protein acting as a sensor of extracellular sodium ion level in a host cell will suffice. Examples of the expression system include expression systems derived from chromosome, episome and virus, for example, vectors derived from bacterial plasmid, yeast plasmid, papovavirus like SV40, vaccinia virus, adenovirus, chicken pox virus, pseudorabies virus, or retrovirus, vectors derived from bacteriophage, transposon, and the combination of these, for instance, vectors derived from genetic factors of plasmid and of bacteriophage such as cosmid or phagemid. These expression systems may contain regulatory sequence that acts not only as a promoter but also as a controller of expressions.

[0039] A host cell that contains the above-mentioned expression system, cell membrane of said host cell, and a protein acting as a sensor of extracellular sodium ion level which is obtainable by the cultivation of said host cell can be used in the screening method of the present invention as hereinafter described. For example, the method of F. Pietri-Rouxel et al. (Eur. J. Biochem., 247, 1174-1179, 1997) or the like can be used as the method to obtain cell membranes, and publicly known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography, preferably high-speed liquid chromatography can be used to pick up said protein acting as a sensor of extracellular sodium ion level from cell cultured material and purify it. As columns used for affinity chromatography, in particular, there are columns to which an antibody to a protein acting as a sensor of extracellular sodium ion level such as an anti-Na_(v)2 monoclonal antibody and the like is bound, or in case that normal peptide tag is added to said protein such as Na_(v)2 or the like acting as a sensor of extracellular sodium ion level, there are columns to which materials having affinity to the peptide tag are bound. These proteins acting as sensors of extracellular sodium ion level can be obtained by using these columns.

[0040] In the present invention, transgenic non-human animal that excessively expresses a gene that codes for a protein acting as a sensor of extracellular sodium ion level is defined as non-human animal that produces larger amount of a protein acting as a sensor of extracellular sodium ion level than wild-type non-human animal does. Though concrete examples of a non-human animal of the present invention include rodents, such as rabbits, mice, rats and the like, a non-human animal of the present invention is not limited to these animals.

[0041] Homozygous non-human animals generated according to Mendelian ratio include excessive expression type that excessively expresses a protein acting as a sensor of extracellular sodium ion level and the littermate wild-type, and it is possible to carry out precise comparative experiments in individual level by using the excessive expression types and the littermate wild-types of these homozygous non-human animals at the same time. Therefore, it is preferable to use animals of the same species, more preferably the littermates, as the wild-type non-human animals, in other words, the non-human animals that excessively express a gene that codes for a protein acting as a sensor of extracellular sodium ion level together in, for example, the screening hereinafter described in the present invention. The generating method of the non-human animals that excessively express a gene that codes for a protein acting as a sensor of extracellular sodium ion level will be explained below, with an example of transgenic mice of a protein acting as a sensor of extracellular sodium ion level.

[0042] The transgenic mice of a protein acting as a sensor of extracellular sodium ion level can be generated in following procedures. A transgene is constructed by fusing promoters such as chicken β-actin, mouse neurofilament, SV40 or the like, and poly A or introns such as rabbit β-globin, SV40 or the like with cDNA that codes for a protein such as Na_(v)2 or the like acting as a sensor of extracellular sodium ion level. The transgene is microinjected in the pronucleus of a fertilized egg of a mouse, and the egg cell is cultured, then transplanted to the oviduct of a recipient mouse. After rearing up the recipient animal, baby mice that have the above-mentioned cDNA are selected from the mice born from the recipient animal. Thus transgenic mice can be generated. The baby mouse that has cDNA can be selected by extracting crude DNA from a tail or the like of a mouse, then carrying out methods like dot hybridization using a gene that codes for a protein acting as a sensor of extracellular sodium ion level as a probe, PCR method using a specific primer and the like.

[0043] By using a protein acting as a sensor of extracellular sodium ion level, a gene that codes for said protein, a fusion protein created by combining a protein acting as a sensor of extracellular sodium ion level and a marker protein and/or a peptide tag, an antibody to a protein acting as a sensor of extracellular sodium ion level, a host cell which contains an expression system that can express a protein acting as a sensor of extracellular sodium ion level, a non-human animal which excessively expresses a gene that codes for a protein acting as a sensor of extracellular sodium ion level, a null mutant non-human animal showing salt intake behavior similar to that of the wild-type animals under water-sufficient conditions and showing much more intakes of hypertonic saline compared with the wild-type animals under water- and salt-depleted conditions, a cell that expresses a protein acting as a sensor of extracellular sodium ion level, or the like, it becomes possible to screen a material that promotes or suppresses the function of a protein acting as a sensor of extracellular sodium ion level or a material that promotes or suppresses the expression of a protein acting as a sensor of extracellular sodium ion level. What obtained by this screening could be a suppressant, a preventive, or a remedy for chronic diseases of human resulted from excessive intake of salt, or substances useful for diagnosis and cure of the diseases resulted from deficiency or abnormality of a protein acting as a sensor of extracellular sodium ion level or the like.

[0044] Examples of said screening methods include a method with cells that express a protein acting as a sensor of extracellular sodium ion level and a subject material; a method with null mutant non-human animals of the present invention showing salt intake behavior similar to that of the wild-type animals under water-sufficient conditions and showing much more intakes of hypertonic saline compared with the wild-type animals under water- and salt-depleted conditions or transgenic non-human animals which excessively express a gene that codes for a protein acting as a sensor of extracellular sodium ion level, and the subject material.

[0045] As a screening method with cells that express a protein acting as a sensor of extracellular sodium ion level and a subject material is, a method wherein cells that express a protein acting as a sensor of extracellular sodium ion level and a subject material are brought into contact, and then the change of the function or the expression of a protein acting as a sensor of extracellular sodium ion level is measured/evaluated is exemplified.

[0046] Concrete examples of a screening method with the null mutant non-human animals showing salt intake behavior similar to that of the wild-type animals under water-sufficient conditions and showing much more intakes of hypertonic saline compared with the wild-type animals under water- and salt-depleted conditions or the transgenic non-human animals which excessively express a gene that codes for a protein acting as a sensor of extracellular sodium ion level and the subject material include; a method wherein nerve cells obtained from the null mutant non-human animals showing salt intake behavior similar to that of the wild-type animals under water-sufficient conditions and showing much more intakes of hypertonic saline compared with the wild-type animals under water- and salt-depleted conditions, or from the transgenic non-human animals which excessively express a gene that codes for a protein acting as a sensor of extracellular sodium ion level are brought into contact with a subject material in vitro, and then the change of the function or the expression of said protein is measured/evaluated; a method wherein saline is administrated to said null mutant non-human animals or to said transgenic non-human animals beforehand, then the nerve cells obtained from said non-human animals are cultured in the presence of the subject material, and the change of the function or the expression of said protein is measured/evaluated; a method wherein saline and a subject material are administrated to said null mutant non-human animals or to said transgenic non-human animals beforehand, then the change of the function or the expression of said protein in the nerve cells obtained from said non-human animals is measured/evaluated; a method wherein saline and a subject material are administrated to said null mutant non-human animals or to the transgenic non-human animals beforehand, then the change of the function or the expression of said protein in the non-human animals is measured/evaluated.

[0047] In measuring and evaluating the change of the function or the expression of a protein acting as a sensor of extracellular sodium ion level, it is preferable to evaluate/compare with the measured value of the wild-type non-human animals, in particular, of the littermate wild-type non-human animals, as control, because it offsets the dispersion caused by the difference between individuals. The function of a protein acting as a sensor of extracellular sodium ion level is to regulate sodium level of body fluid in vivo, that is, to act as a sensor of extracellular sodium ion level. Although concrete examples of the change of said function include the change of the function in the sensory circuits for body fluid osmolarity, the change in preference-aversion response to the intake of water and salt and the like, the change of the function is not limited to these examples.

[0048] The medical compounds of the present invention are not limited particularly as long as the medical compounds contain said protein of the present invention that acts as a sensor of extracellular sodium ion level, or materials that promote or suppress the function of a protein acting as a sensor of extracellular sodium ion level, or materials that promote or suppress the expression of a protein acting as a sensor of extracellular sodium ion level as their effective components, and these medical compounds can be used for the treatment of the patients who need promotion of the function or enhancement of the expression of a protein acting as a sensor of extracellular sodium ion level, or the patients who need suppression of the function or the expression of a protein acting as a sensor of extracellular sodium ion level.

[0049] The present invention will be explained in detail with embodiments below, but the technological scope of the present invention is not limited to these embodiments.

[0050] [Construction of a Targeting Vector]

[0051] In order to construct a targeting vector, genomic fragments of mice containing protein-coding exons 1, 2 and 3 were cloned by NaG/SCL11 probe of rats. Nine independent genomic clones were isolated from mouse genomic libraries (donated by Dr. Mori, Osaka Univ., Japan) prepared from a cell line R1, by hybridization with a 446-bp fragment of rat NaG cDNA (nucleotide residues 11-456 encompassing the first three coding exons: GenBank accession number Y09164: Seq. ID No. 1). By Southern blot analysis using several restriction enzymes, it has been confirmed that all these overlapping clones were derived from a single genomic locus. The hybridization-positive 3.2 and 3.7 kb HindIII fragments were subcloned into pBluescript II SK (−) (Stratagene) and subjected to DNA sequencing (the sequence is in GenBank under accession number AF190472: Seq. ID No. 4). The 3.2 kb fragment contained protein-coding exon 1 (13 bases of the 5′ untranslated region and the first 238 bases in the mouse Na_(v)2 protein-coding sequence: nucleotide residues 238-490 of GenBank accession number L36179) and the 3.7 kb fragment contained exons 2 (nucleotide residues 491-609) and 3 (nucleotide residues 610-701). The DNA sequence encoding the three exons was identical to that of mouse Na_(v)2.3 CDNA cloned by Tamkun and coworkers (J. Biol. Chem. 269, 30125-30131(1994)). The cloned genomic fragments showed identical restriction maps with mouse genomic DNA on Southern blot analysis with four restriction enzymes (BamH I, Bgl II, EcoR I and Hind III). This finding indicates that mouse Na_(v)2.3 is the species counterpart of rat NaG. Accordingly, Na_(v)2.3, NaG and SCL11 are designated as Na_(v)2.

[0052] In order to construct the targeting vector, the lacZ gene was so designed that it would be inserted into the protein-coding exon 1 of the mouse Na_(v)2 gene and that the N-terminal twenty amino-acid sequence of mouse Na_(v)2 would be fused with β-galactosidase. In other words, the 12.5 kb Sal I fragment containing the three exons was inserted into the Xho I site of pDT-A (Anal. Biochem. 214, 77-86 (1993)), then the Sal I-Xho I fragment of the lacZ-neo cassette was introduced into the endogenous Xho I site located in exon 1 (see FIG. 1a). This results in the expression of a protein that fuses the first 20 amino acids of the mouse Na_(v)2 protein with the N-terminus of β-galactosidase. The original genomic structure of mouse Na_(v)2 gene was not modified except for the insertion of the lacZ-neo cassette to make sure that the lacZ gene is expressed in place of the mouse Na_(v)2 gene in the targeted mice. An EcoR I linker was inserted beforehand into the 5′ terminus of the lacZ-neo cassette to utilize it as an exogenous restriction site in Southern blot screening analysis. FIG. 1a shows restriction maps of the targeting vector (top), mouse Na_(v)2 gene locus of wild-types (middle), and recombinant gene locus (bottom). In FIG. 1a, restriction sites B, Bg, E, H, and X represent BamH I, Bgl II, EcoR I, Hind III and Xho I respectively.

[0053] [Generation of Na_(v)2 Knockout Mice]

[0054] The above-mentioned filamentous shaped targeting vector was induced into ES cells (R1 cell line derived form 129/SV mouse) by an electroporation method. According to the method described in the paper (Neurosci. Lett. 247, 135-138 (1998)), neomycin-resistant ES clones were selected by G418, and screening of the targeted clones were performed. Homologous recombination was verified by Southern blot analysis using EcoR I digestion with probe 1 (see FIG. 1a; the 0.3 kb EcoR I-Xba I fragment located about 8 kb upstream from the Xho I site in exon 1). The selected clones were checked using probe 2 (the 0.6 kb Pst I fragment derived from the neo gene). The targeted locus was verified by genomic PCR using one sense primer (primer 1, ATGTTGACTTCCCCAGAGCC in the 5′-terminal region of exon 1, Seq. ID No. 5) and two antisense primers (primer 2, AACCAGGCAAAGCGCCATTC in the 5′-terminal region of lacZ, Seq. ID No. 6: primer 3, CATCTTCCAAGGGCTCTGACA in the 3′-terminal region of exon 1, Seq. ID No. 7). PCR amplification was carried out in two stages with EX-Taq polymerase (Takara) according to the manufacturer's protocol using a programmable thermal cycler (first stage; 95° C. for 5 minutes, 60° C. for 1 minute, 72° C. for 1 minute: second stage; 95° C. for 30 seconds, 60° C. for 30 seconds, 72° C. for 1 minute).

[0055] Two out of 98 ES clones, which were identified to have the correct homologous recombinant allele, were used for injection into eight-cell stage C57BL/6J mouse embryos. The injected embryos were cultured in M16 medium overnight up to blastocysts, and seven to ten blastocysts were transplanted into the uterus of a recipient ICR mouse. The male chimeric mice thus obtained were mated with C57BL/6J females, and heterozygous mice (F1: first filial generation) were generated from littermates, and this heterozygous mutant mice were intercrossed subsequently in order to obtain homozygous mice, then Na_(v)2 deficient mice, which were produced according to Mendelian ratio, were generated.

[0056] The null mutant animals (mouse Na_(v)2^((−/−)) were healthy, fertile and apparently normal. The genotypic analysis of 458 four-week-old offspring obtained from breeding heterozygous animals showed an approximately Mendelian ratio between wild-type (29.5%, n=135), heterozygous mutant (48.2%, n=221) and homozygous mutant (22.3%, n=102) animals. This suggests that embryonic development and body functions were not significantly impaired in either heterozygous or homozygous mutant animals. It was reported that acute and transient expression of mouse Na_(v)2 was induced perinatally to the uterine smooth muscle (J. Biol. Chem. 269, 30125-30131 (1994), Am. J. Physiol. 270, C688-696 (1996)). It should be noted that pups were delivered normally in Nav2^(−/−)mice. The pattern of lacZ expression in the null mutants was identical to that in the heterozygous mutants except for the intensity of the expression, suggesting that deficiency of mouse Na_(v)2 does not affect the differentiation or viability of the mouse Na_(v)2-expressing cells.

[0057]FIG. 1b shows the result of Southern blot analysis in which samples of genomic DNA were derived from tails of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice, and then membranes blotted with genomic DNA digested with EcoR I were hybridized with said probe 1 located on the 5′ side of the targeting vector. The sizes for the wild-type (18 kb) and recombinant (10 kb) genotypes are shown on the right of FIG. 1b. FIG. 1c shows the result of genomic PCR analysis of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mutant mice. The sizes for the wild-type (200 bp) and recombinant (400 bp) genotypes are shown on the right of FIG. 1c.

[0058] [Confirmation of Non-expression of Na_(v)2 in Na_(v)2 Knockout Mice]

[0059] The mouse Na_(v)2 protein expression in the mutant mice was examined by Western blot analysis. Lung tissue samples of each wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice were prepared according to the methods of Knittle et al. (Am. J. Physiol. 270, C688-696 (1996)). SDS-polyacrylamide gel electrophoresis and immunoblotting were carried out according to the method described in the paper (Neurosci. Lett. 247, 135-138 (1998)). Anti-mouse-Na_(v)2.3 antiserum (a generous gift from Dr. M. Tumkun, Colorado State Univ., Colo.) was used at a 1:500 dilution in PBS. The immunoblots were incubated with several non-immune sera to verify that it was not non-specific combination. The results are shown in FIG. 1d. The position of Na_(v)2 protein (220 kDa) is indicated on the right of FIG. 1d. The sodium channel gave a broad signal, since Na_(v)2 protein is highly glycosilated and readily aggregates even in the SDS-containing buffer. Compared with wild-type mice, about a half of the amount of mouse Na_(v)2 protein in heterozygous mutant (Na_(v)2^(+/−)) mice, and no mouse Na_(v)2 protein in homozygous mutant (Na_(v)2^(−/−)) mice was detected in lung membrane preparations. It is indicated that the allele was a null mutation because mouse Na_(v)2 protein was not expressed.

[0060] [Confirmation of the Expression of lacZ in Na_(v)2 Knockout Mice by X-Gal Staining]

[0061] Embryos were fixed by immersion in 3.5% formaldehyde in PBS for 1 hour at room temperature, and then cut midsagittally with a razor. FIG. 2a shows X-Gal staining of a whole-mount mouse Na_(v)2+/−embryo at embryonic day 15 (E15). Intensive β-galactosidase activity was observed in the trigeminal ganglia (pointed by the arrowhead in FIG. 2a) and dorsal root ganglia (pointed by the arrow in FIG. 2a)(see reference photograph 1). In this E15 mouse, lacZ-expression was evident also in the lung (pointed by the asterisk). In these organs, the expression of lacZ persisted into adulthood. When dorsal root ganglia were cut into thin sections after X-Gal staining, β-galactosidase activity was detected in spinal sensory neurons with various cellular diameters. An X-Gal stained cryostat tissue section of dorsal root ganglion of postnatal day 2 Na_(v)2^(+/−)mouse was shown in FIG. 2b (see reference photograph 1). The expression of lacZ was confined to the somata of neurons within dorsal root ganglion (nerve tracts are shown by asterisk in FIG. 2b), and not detected in axons. A similar pattern of lacZ expression was observed in tissue sections of the trigeminal ganglia. A cryostat section of adult sympathetic nerve trunk in the thoracic region is shown in FIG. 2c (see reference photograph 1). Based on the appearance, distribution, and size of the cell bodies, the numerous intensely stained cells are likely to be Schwann cells. The expression of lacZ was also observed in cardiac autonomic nerves and lingual nerves. These patterns of lacZ expression agreed well with the results of rat Na_(v)2 and mouse Na_(v)2 expressions (Proc. Natl. Acad. Sci. USA 89, 7272-7276 (1992), FEBSLett. 400, 183-187 (1997), Glia, 21, 269-276 (1997)). This indicates that the lacZ gene expression is duly under the control of the regulatory regions of mouse Na_(v)2 gene. In FIG. 2c, the arrowheads identify the somata of Schwann cells, and scale bar =50 μm.

[0062] [Physiological Roles of Na_(v)2]

[0063] In order to examine the physiological roles of mouse Na_(v)2, the lacZ-expression was surveyed throughout central nervous system (CNS) using brains of Na_(v)2^(+/−)and Na_(v)2^(−/−)mice. Postnatal animals were perfused under pentobarbital anesthesia first with PBS and then with the fixative. The fixed brains were cut coronally at 2 mm thick or sagittally with a razor. Samples were rinsed twice with PBS and incubated overnight in PBS containing 1 mg/ml X-Gal, 5 mM K₃Fe(Cn)₆, 5 mM k₄Fe(CN)₆, 2 mMMgCl₂, and 0.2% NP-40 at 37° C. For immunostaining, some X-Gal stained slices were cut further into coronal sections at 14 μm thick with a cryostat microtome and mounted onto gelatin-coated slides. With rabbit polyclonal antibodies to anti-neurofilament 200 (Sigma, N-4142) or to anti-glial fibrillary acidic protein (GFAP) (Santa Cruz Biochemistry, sc-6170), immunostaining was performed (Neurosci. Lett. 247, 135-138 (1998)). FIG. 3 shows that mouse Na_(v)2 was expressed in specialized neurons and ependymal cells in the adult CNS (see reference photograph 2).

[0064] In FIG. 3, lacZ expression in the CNS of Na_(v)2^(+/−)(FIG. 3a-e) and Na_(v)2^(−/−)(FIG. 3f) mutant mice were shown. Fixed adult brains were cut coronally (FIG. 3a, b, d, e and f) or midsagittally (FIG. 3c) at 2 mm and then stained with X-Gal. In (FIG. 3c), the skull under the brain was not removed. In (FIG. 3e), homozygous mutant mice were used for the analysis to detect the locus of low level expression. Explanations of the abbreviations in FIG. 3 are as follows: AH, anterior hypothalamic area; MH, medial habenular nucleus; ME, median eminence; OVLT, organum vasculosum laminae terminalis; MPO, medial preoptic area; DMH, dorsomedial hypothalamus; IPDM, interpeduncular nucleus of the dorsomedial part; MMR, medial part of the median raphe; NHP, neurohypophysis; SFO, subfornical organ; CX, cerebral cortex; BLA, basolateral amygdala. In (FIG. 3c), OVLT was removed from the central nervous system and attached to the skull. The coronal semi-whole-mount brains were cut 50 μm thick using cryostat microtome and then stained with anti-neurofilament polyclonal antibodies (FIG. 3g and h), anti-GFAP polyclonal antibodies (FIG. 3i), or cresyl violet (FIG. 3j). Brown signals are the site that reacted with the antibodies. The samples are AH (FIG. 3g), SFO (FIG. 3h and i) and ME (FIG. 3j). Arrowheads indicate double positive neurons. The asterisk in FIG. 3j indicates the third ventricle. The dorsal side is toward the top of the panels. Scale bar: FIG. 3g to FIG. 3h=30 μm; FIG. 3j=100 μm.

[0065] As shown in FIG. 3, clusters of lacZ expression were limited to specific loci in the central nervous system (FIG. 3a-f): MPO, AH, DMH, IPDM, MMR, MeV, MH, ME, SFO, OVLT and NHP. ME, SFO, OVLT and NHP are known as the circumventricular organs (CVOs) having unusual dense and permeable capillary networks that facilitate secretion of substances into blood or penetration of substances into important tissue (FASEB J, 7, 678-686 (1993)). Relatively weak lacZ expression was detected in CX and BLA in Na_(v)2^(+/−)mice. The intensity of the lacZ expression in these areas was more evident in Na_(v)2^(−/−)mice (FIG. 3f). To examine the cell-types expressing lacZ, the brains were stained with X-Gal and cut into tissue sections with a cryostat microtome and subsequently immunostained with anti-neurofilament polyclonal antibodies or anti-glial fibrillary acidic protein (GFAP) polyclonal antibodies, or stained with cresyl violet. Most of the cells expressing lacZ were positive for neurofilament in the MPO, AH (FIG. 3g), IPDM, MMR, MH and MeV. GFAP-positive cells were negative for the lacZ expression, suggesting that astrocytes are negative for mouse Na_(v)2.

[0066] The distribution of lacZ-expressing cells in the CVOs was of particular interest. In the ME, the lacZ-expressing cells lined the floor of the third ventricle (FIG. 3j). This distribution corresponds to the location of nonciliated ependymal cells. These cells are thought to be tanycytes, which are characteristic cells providing a morphological connection between cerebrospinal fluid (CSF), nerve cells and blood vessels (Neuroscience 3, 277-283 (1978)). They are thought to be involved in exchange of substances between the CSF and pericapillary space. The lacZ-positive cells were sparsely distributed all over the SFO, and most of them were co-localized with neurofilaments (FIG. 3h) and negative for GFAP (FIG. 3i). Intensive lacZ-positive cells also populated lining the entire third ventricle, suggesting that they are ependymal cells. In the NHP, the X-Gal signals were densely clustered. They are likely to correspond to so-called pituicytes (J. Exp. Biol. 139, 67-79 (1988)).

[0067] [Fos-immunohistochemistry]

[0068] Since analysis of the lacZ expression clearly demonstrated that mouse Na_(v)2 was expressed in the four CVOs and several nuclei in the central nervous system and that the mouse Na_(v)2-expressing cells were thus diverse not only in tissue distribution but also in cell types, it became difficult to obtain a unified view of channel function or property. However, the four CVOs are thought to be involved in body-fluidhomeostasis (FASEBJ, 7, 678-686 (1993), Annu. Rev. Physiol. 59, 601-619 (1997), Physiol. Rev. 78, 583-686 (1997), Physiol. Rev. 58, 582-603 (1978), Ann. NY Acad. Sci, 877, 258-280 (1999)). If the mouse Na_(v)2 channel functions in the sensory circuits for body-fluid osmolarity, it was expected that the activity and gene expression in these organs would be affected in the mouse Na_(v)2 mutant mice. Therefore, the effects of water deprivation on the central expression of Fos, a nuclear protein and a marker of changes in neural activity in response to the extracellular fluid balance in mice and rats, were examined as follows.

[0069] The time course of changes in Fos-immunopositive cell density in five regions of the brain (the medial preoptic nucleus (MnPO), organum vasculosum laminae terminalis (OVLT), subfornical organ (SFO), paraventricular nucleus (PVN), and supra optic nucleus (SON)) under water-sufficient and water-depleted conditions were examined with mice which were deprived of water for 0 h (n=4 for mouse Na_(v)2^(+/+), n=4 for mouse Na_(v)2^(−/−)), 12 h (n=5 and 5), 24 h (n=6 and 7), or 48 h (n=6 and 5). The mice were perfused with the fixative as described above, and their brains were immersed in the same fixative at 4° C. overnight. Brains were cut coronally into sections at 50 μm thick on a vibratome (Leica, VT1000S). Immunostaining was performed with a goat anti-Fos polyclonal antibody (Santa Cruz Biochemistry, sc-52-G) at a dilution of 1:1000 in PBS. Sections containing regions of interest were chosen and the Fos-immunopositive nuclei were enumerated. Each area was measured by using an image analysis system (KS400 attached to Axiophoto 2). The number of nuclei present per mm² was determined in the above-mentioned five regions of the brain. The results are shown in FIG. 4 (see reference photograph 3).

[0070]FIG. 4a is typical examples of tissue sections containing the OVLT derived from wild-type (+/+) and null mutant (−/−) mice under water-sufficient or 24-h dehydrated condition. Scale bar=200 μm. In FIG. 4b, mean numbers of Fos-immunopositive cells per mm² in subfornical organ (SFO), supraoptic nucleus (SON), paraventricular nucleus (PVN), organum vasculosum laminae terminalis (OVLT) and the median preoptic nucleus (MnPO) during water deprivation were plotted. The vertical bars indicate S. E., the asterisk shows that a significant difference (P<0.05) between Na_(v)2^(−/−)and Na_(v)2^(+/+)mice were revealed. In the water-sufficient condition, Fos-immunopositive cells were not detected in any region examined. At 12, 24 and 48 h after water deprivation, number of cells with Fos-immunopositive nuclei was remarkably increased in these regions in both Na_(v)2^(−/−)and Na_(v)2^(+/+)mice. However, in SFO and OVLT, approximately two-fold increases in Fos-immunopositive nuclei were observed in Na_(v)2^(−/−)mice as compared with in Na_(v)2^(+/+)mice. In the MnPO, PVN and SON, on the other hand, the rates of increase in Fos-immunopositive cells were comparable between the two groups.

[0071] [Behavioral Analysis]

[0072] The effect of mouse Na_(v)2-channel deficiency on water and salt intakes of mice was next examined. For the behavioral study, the mutant mice were backcrossed with C57 BL/6J males. As a result, it was verified that the behavior of F1 and F4 mice was identical. The preferance-aversion behavior of the homozygous-, heterozygous-mutant and wild-type littermates were measured by a 48-h two-bottle preference test. Mice were presented with a choice between distilled water and a tasting solution for 48 h in their home cage. For all behavioral studies, male mice at 12-24 weeks of age were used. They are individually housed under constant room temperature, humidity and 12/12 h light-dark cycle. The positions of the two bottles were switched every 24 h to avoid side preference. The total intake for each animal was measured and used to calculate a preference ratio according to the following formula: preference ratio=volume of tasting solution (ml)/total intake volume of tasting solution and water (ml). The results are shown in FIG. 5. Preference ratios for NaCl solutions with a series of concentrations are shown in FIG. 5a, and that for three fundamental tastants with fixed concentrations are shown in FIG. 5b. Homozygous (−/−), heterozygous (+/−) and wild-type (+/+) mice, five mice each, were used here. FIG. 5a shows that the concentration sensitivity to a series of NaCl solutions was comparable among the three groups of mice under the condition satiated with water and salt, and all the groups showed maximum preference to 0.1 M NaCl and evasiveness to 0.3 M or higher concentration of NaCl. The null mutants showed normal preferences to various tastants under the condition satiated with water and salt. FIG. 5b shows that preference ratios to sweet (0.5 M sucrose), sour (0.01 M HC1) and bitter (0.02 M quinine hydrochloride) tasters were not different among the groups.

[0073] [Electrophysiology]

[0074] To verify the normality in taste responses in the null mutants, electrophysiological analysis was performed on the chorda tympani nerve, which is known to be the nerve fiber responsible for tasting NaCl. Male mice at 12-24 weeks of age were used (4 wild-type and 5 homozygous mice for the normal condition; 3 wild-type and 5 homozygous mice for the acute salt-appetite condition). Each mouse was deeply anesthetized by an intraperitoneal injection of sodium pentobarbital (60 mg/kg), then tracheotomized and secured with a head holder. The chorda tympani nerve was exposed, freed from surrounding tissues, and cut at the point of its entry to the bulla. The whole bundle of the nerve was dissected and lifted on a platinum wire recording electrode (0.1 mm in diameter). An indifferent electrode was attached to nearby tissue. The nerve activities were amplified, displayed on an oscilloscope, and monitored with an audioamplifier. The amplified signal was passed through an integrator with a time constant of 0.3 sec and was displayed on a slipchart recorder.

[0075] The taste solutions were 0.1 M NH₄Cl, 0.1 M NaCl, 0.5 M sucrose, 0.01 M HCl, 0.02 M Q-HCl, 0.1 M KCl and 0.1 M CH₃COONa (AcNa). These solutions were made up with distilled water and 0.1 mM amiloride solution. Each solution and rinsing water were applied to the anterior part of the tongue at room temperature (25±2° C.). The tongue was rinsed for at least 45 sec between successive stimuli. The magnitude of the whole nerve response was measured as the height of the integrated response from the baseline at 10 sec after onset of stimulation. Recorded results of the chorda tympani response to various taste stimuli are shown in FIG. 6a. The magnitudes of the responses to various taste stimuli expressed as relative values with the magnitude of the response of 0.1 M NH₄Cl taken as the standard are shown in FIG. 6b.

[0076] The neurophysiological responses to 0.02 M Q-HCl, and 0.1 M CH₃COONa were of similar intensity between the null-mutant and wild-type mice. Responses to 0.1 M NaCl and 0.1 M CH₃COONa were decreased to the same degree by amiloride application in both groups of mice, indicating that amiloride-sensitive channels in taste buds in the mutant mice function normally. Similar results were observed in the null-mutant and wild-type mice under the acute salt appetite condition. This finding together with the normal behavioral response to various tastants under water- and salt-sufficient conditions (FIG. 5) shows that the taste reception of the null mutants is not impaired.

[0077] Under the water-depleted condition, animals take in a large quantity of water and avoid hypertonic saline to recover from the hypertonic state. The preference to hypertonic saline (0.3 m NaCl) before and after 24-h dehydration was examined. Before testing, mice were trained to drink water from two bottles for one week. On the day before dehydration, mice were presented with a choice between water and 0.3 M NaCl at 10.00 h, and then measured for fluid intake at 16.00 h. At 10.00 h on the next day, the bottles were removed. Dry food was placed throughout the period of water deprivation. After 24-h dehydration, the two bottles were returned and fluid intakes were measured at 16.00 h. The results are shown in FIG. 7. The null mutants showed an abnormal ingestion of hypertonic saline under the water-depleted condition. Preference ratio for 0.3 M NaCl solution (FIG. 7a) and total fluid intake (FIG. 7b) were measured before and after 24-h dehydration. In this experiment, n=6(+/+), 6(+/−) and 6(−/−). Vertical bars in the FIG. 7 indicate S. E., and the asterisk shows that a significant difference (P<0.05) between Na_(v)2^(−/−)and Na_(v)2^(+/+)mice were observed. In contrast to the wild-type and heterozygous mutant mice, which showed markedly decreased preference ratios to hypertonic saline after dehydration, the null mutants showed no change in the preference ratio (FIG. 7a). Total water intake (water plus 0.3 M NaCl) did not differ among the groups both before and after 24-h dehydration. The total water intake of all the groups showed more than a four-fold increase after dehydration (FIG. 7b).

[0078] Blood was recovered from animals before or after dehydration by decapitation, and then the concentrations of plasma electrolytes were measured by using an electrolyte analyzer (9180, AVL Scientific, GA). The electrolyte concentrations in the serum before and after dehydration were normal in both wild-type and homozygous mutant mice (n=6 each). The electrolyte concentrations in wild-type and homozygous mutant mice before dehydration are as follows respectively: 153.6±0.6 and 153.0±1.2 mM for Na⁺; 4.6±0.1 and 4.7±0.1 mM for K⁺; 118.5±0.6 and 118.3±0.9 mM for Cl⁻. The electrolyte concentrations in wild-type and homozygous mutant mice after dehydration are as follows respectively: 151.6±0.8 and 150+0.3 mM for Na⁺; 6.5±0.2 and 6.7±0.2 mM for K⁺; 116.0±1.0 and 116.4 ±0.8 mM for Cl⁻. This suggests that the null mutants immediately excreted excessive amounts of sodium into urine, and thus the renal function of null mutants works normally.

[0079] Furthermore, motivated salt appetite was induced by intraperitoneal injection of a diuretic drug, furosemide, and sodium-depleted food, then the sodium-depletion induced salt appetite test was carried out in the following way. Before testing, control measurements of water and 0.3 M NaCl intake were performed for several days. At 10.00 h, mice were injected intraperitoneally with 0.12 ml of normal saline (0.9% NaCl). The bottle of 0.3 M NaCl was withdrawn and sodium-depleted food was supplied in place of a normal diet. The second injection of normal saline was given at 16.00 h. On the following day, water and 0.3 M NaCl were presented at 10.00 h and intakes of 0.3 M NaCl and water were measured at 12.00, 14.00 and 16.00 h. After that, a similar protocol with furosemide injection (0.6 mg in 0.12 ml of normal saline) was performed with sodium-depleted food in the same mice (acute salt-appetite condition). Finally, the same protocol, except that normal sodium-containing food was supplied, was performed to evaluate the effect of sodium-depleted food. The results are shown in FIG. 8.

[0080] The ingested volumes of water and 0.3 M NaCl are shown in FIG. 8 in cumulative values for every 2 h. The results of the behavioral study are shown in FIG. 8 as plotted mean cumulative intakes of 0.3 M NaCl (right) and water (left) per 2 h starting just after each experimental procedure as follows: a sodium-depleted diet combined with normal saline injection (top), a sodium-depleted diet combined with furosemide injection (middle), and a sodium-contained diet combined with furosemide injection (bottom). In this experiment, n=10 (+/+), 10 (+/−) and 10 (−/−). Vertical bars in FIG. 8 indicate S. E., and the asterisk shows that a significant difference (P<0.05) between Na_(v)2^(−/−)and Na_(v)2^(+/+)mice were observed. As shown in FIG. 8, under the control condition in which isotonic saline was injected in place of a furosemide solution, the ingested volumes of water and 0.3 M NaCl were comparable among the three groups (graphs at the top). Under the acute salt appetite condition induced by furosemide injection with a sodium-depleted diet, however, the null-mutants showed an approximately two-fold increase in the ingestion of 0.3 M NaCl compared with the wild-type and heterozygous mutant mice (right graph at the middle). This abnormal ingestion of hypertonic saline stopped when sodium-containing food was provided (graph at the bottom).

[0081] [Na_(v)2 Channel is a Sodium Concentration-dependent Sodium Channel]

[0082] It has been verified that Na_(v)2 channel is a sodium concentration-dependent sodium channel by following experiments.

[0083] First, an anti-Na_(v)2 antibody was constructed as follows: a peptide comprised of an amino acid sequence (SVSETVPIASGESDIK; Seq. ID No. 8), which exists in inter domain 2-3 of rat Na_(v)2 channel, was combined to hemocyanin, then a white rabbit was immunized with the hemocyanin-combined peptide as an immunogen, and anti-rat Na_(v)2 rabbit antiserum was constructed by a usual method. The constructed antiserum was purified by a column in which the peptide used as the immunogen was fixed, and an anti-Na_(v)2 antibody was obtained. The specificity of the anti-Na_(v)2 antibody was confirmed by Western blot and immunohistochemical analysis. When the purified anti-Na_(v)2 antibody was used, no non-specific positive signal was observed in sections of brain, lung, dorsal root ganglia and tongue prepared from gene-deficient mice.

[0084] Next, nerve cells in dorsal root ganglia were isolated. The dorsal root ganglia were prepared from wild-type and Na_(v)2 gene-deficient mice of 8-16 weeks of age. Nerve cells were dispersedly isolated from the dorsal root ganglia according to the method of Renganathan et al. (J Newrophysiol 84, 710-718, 2000). Before used for an ion imaging experiment, the dispersedly isolated nerve cells were cultured under the condition of the humidity of 100% and the temperature of 37° C., and with 5% of carbon dioxide, then adhered to the glass of culture plates. All nerve cells were confirmed to be Na_(v)2-positive by staining nerve cells of dorsal root ganglia derived from wild-type mice with the above-mentioned anti-Na_(v)2 antibody. The size of the dispersedly isolated nerve cells were comprised of 3 groups of small (25 micron or smaller in diameter: about 50%), medium (25 to 40 micron in diameter: about 40%), and large (40 micron or larger in diameter: about 10%). However, there was no difference between the materials isolated from wild-type and gene-deficient mice in the size, shape and survival rate of these 3 types of cell. The survival rate was verified by Tripan blue staining.

[0085] In addition, nerve cells of subfornical organs were also isolated. The subfornical organs were prepared from wild-type and Na_(v)2 gene-deficient mice of 8-16 weeks of age. In order to visualize the subfornical organs, Evans blue was intraperitoneally injected in advance. The subfornical organs were dispersed according to the method of Jurzak et al. (Brain Res 662, 198-208, 1994). As in the case of nerve cells of dorsal root ganglia, the dispersedly isolated nerve cells were cultured under the condition of the humidity of 100% and the temperature of 370 C, and with 5% of carbon dioxide, then adhered to the glass of culture plates, and then used for the experiment. Some nerve cells of subfornical organs derived from wild-type mice were stained with the above-mentioned anti-Na_(v)2 antibody, and a ratio of Na_(v)2-positive nerve cells was about 20 to 30%. There was no difference between the nerve cells isolated from wild-type and gene-deficient mice in the size and survival rate.

[0086] With regard to the nerve cells of dorsal root ganglia or subfornical organs prepared from the above-mentioned wild-type and Na_(v)2 gene-deficient mice, intracellular sodium ion and intracellular calcium ion were measured. SBFI/AM (sodium-binding benzofuran isophthalate acetoxymethyl ester) was used for measuring intracellular sodium ion, and Fura-2/AM was used for measuring intracellular calcium ion. Cultured cells loaded with these indicators were adhered to culture plates, and the culture plates were fixed to the stage of a microscope. Fluorescence ratio (F340/F380) was monitored by the fluorescence imaging system. In order to measure data, samplings were conducted every 10 seconds for the nerve cells of dorsal root ganglia, and every 20 seconds for those of subfornical organs respectively. Before the measurement, the nerve cells were incubated with physiological isotonic liquid (145 mM of extracellular sodium concentration) for 30 to 60 minutes, and while measuring, the nerve cells were exposed to a certain perfusate (5 mM of KCl, 2.5 mM of CaCl₂, 1 mM of MgCl₂, 10 mM of HEPES, 10 mM of NaOH, NaCl at the prescribed concentration, pH 7.4, (neutralization by HCl), and the perfusate (extracellular liquid) was perfused at a certain speed (1 ml/min) at room temperature.

[0087] The results of the fluorescence imaging of fluorescence ratio (F340/F380) in nerve cells of dorsal root ganglia are shown in FIG. 9 and FIG. 10. As shown in FIG. 9, when the concentration of the extracellular NaCl was increased to 145 to 170 mM, the concentration of intracellular sodium ion in nerve cells of dorsal root ganglia derived from wild-type mice showed a rapid increase, and reached to steady state straightly. On the other hand, in the nerve cells of dorsal root ganglia derived from gene-deficient mice, an increase of the intracellular sodium ion concentration, such as observed in wild-type mice, was not observed at all (FIGS. 9a and 9 b). Further, the increase of sodium in the nerve cells of dorsal root ganglia derived from wild-type mice was observed in nerve cells of every size (FIG. 9c). This is consistent with the fact that Na_(v)2 expresses in every nerve cells of dorsal root ganglia of wild-type mice.

[0088] In addition, as shown in FIG. 10, the increase of the intracellular sodium ion concentration was caused not by osmotic pressure stimulus using mannitol or by single stimulus of chlorine ions using choline chloride, but by single stimulus of sodium ions using sodium methanesulfonate (FIG. 10a). Accordingly, it has been found that this phenomenon is caused only by the increase of sodium ion concentration. When the extracellular sodium concentrations were arranged to be 120, 130, 140, 150, 160, 165, 170, 180, 190 and 200 mM, 10 concentrations in all, with sodium chloride and the changes in intracellular sodium ion concentration were analyzed, there was no response in the range of 120 to 150 mM, the increase was observed in the extracellular sodium ion concentration of 160 and 165 mM, and extremely significant increase was observed in the extracellular sodium ion concentration of 170 mM and higher (Fig. 10b). In case the extracellular sodium was rearranged to the original concentration of 145 mM at that time, it was observed that the intracellular sodium concentration gradually changed to the original concentration. As there was no intracellular sodium store, it was concluded that this increase of the intracellular sodium concentration was attributed to an influx from extracellular regions via Na_(v)2 channel. The threshold value of the channel opening is presumed to be in the range of 160 to 170 mM.

[0089] In order to investigate the possibility that molecules other than Na_(v)2 channel are involved in the influx of sodium into the cells accompanied with this increase of the extracellular sodium ion concentration, the effects of each blocker of various sodium ion pumps, sodium ion transporters, sodium ion channels were examined. As a result, it was found that there was no influence of TTX-sensitive voltage-dependent sodium channels (Fig. 10c), amiloride-sensitive sodium channels (Fig. 10c), sodium glucose co-transporters, sodium calcium antiporters, sodium potassium chloride transporters and sodium potassium pumps. Further, it was concluded that TTX-nonsensitive voltage-dependent sodium channels were not involved in this phenomenon observed in cell types of every size because they expressed specifically in small nerve cells of dorsal root ganglia. The fact that sodium calcium antiporters were not involved in this phenomenon was also confirmed by calcium imaging (FIG. 10d).

[0090] The results of the fluorescence imaging of fluorescence ratio (F340/F380) in nerve cells of subfornical organs are shown in FIG. 11. As shown in FIG. 11, basically same results as in the case of the nerve cells of dorsal root ganglia are obtained also in the nerve cells of subfornical organs, which are organs for detecting sodium ion concentration in the central nervous system. About 20 to 30% of the nerve cells of subfornical organs were Na_(v)2 immuno-positive cells, and an increase of the intracellular sodium ion concentration was observed in these Na_(v)2 immuno-positive cells, however, no increase of the intracellular sodium ion concentration was observed in Na_(v)2 immuno-negative cells, as in the case of Na_(v)2 gene-deficient mice. These results verify that Na_(v)2 is a sodium channel which makes sodium ion flow into cells in an extracellular sodium ion concentration-dependent manner in subfornical organs as well. In other words, it has been found that Na_(v)2 is a new sodium channel of a sodium concentration-dependent sodium channel.

INDUSTRIAL APPLICABILITY

[0091] A null mutant non-human animal characterized in showing salt intake behavior similar to that of wild-type animals under water-sufficient conditions and showing much more intakes of hypertonic saline compared with wild-type animals under water- and salt-depleted conditions, for example, an Na_(v)2 channel gene-deficient mouse, is useful as a model animal of excessive salt intake experiments. By using this Na_(v)2 channel gene-deficient mouse, it has been revealed that Na_(v)2 channel acts a role to sense and control sodium ion level in cerebrospinal fluid, and that Na_(v)2 channel expresses in neurons and ependymal cells in restricted areas of the central nervous system, particularly in the circumventricular organs which are involved in body-fluid homeostasis, and that Na_(v)2 channel plays an important role in the central sensing of body-fluid sodium level and regulation of salt intake behavior.

Sequence Listing

[0092] <110>JAPAN SCIENCE AND TECHNOLOGY CORPORATION

[0093] <120>Nav2 channel gene-deficient non-human animals

[0094]

1 8 1 446 DNA Rattus norvegicus 1 atgttgactt ccccagagcc gaagggcctt gtcccattca cggcagagtc acttgaactt 60 ataaaaaatc acattgctaa aaaatgcaac gaagagcatg aagaagaaga tttaaaacca 120 agccgggata tagaagcagg caaaaaactt ccatttgcct atggaaccct tcctcaagga 180 accgtgtcag agcccttgga agatgtggat ccatactact atgttaagag aaatactttc 240 atggtcttaa acagaaacag agtcatcttc aggttcaatg cggtttccat cctctgcacg 300 ttgtctcctt taagctctct cagaagagct gttatcaagg ttttggtgca cccccttttg 360 cgcctgctga ttttaattag tgttctcacc gacagcatac ttatgtgcat gagtaaccta 420 ccggaatgga tattggcagt agagaa 446 2 5482 DNA Mus musculus CDS (252)..(5297) 2 cacgcgtcga ctagtacggg ggggggggag gggttggtct gtaggtggtc tctgggtctg 60 tggagctagc ctggtggctg agtgtttagc tggaagcagc agtggaccgc aaccacattg 120 caacaacctc cgtagtagag atctgagaag acaagcccag gagagcaaag ggctctcgtg 180 agccttgcat ctggggttct tgctggagtt ttagtgaaga ctagcatttg acagcaacta 240 taaaaccgaa a atg ttg act tcc cca gag ccg aag ggc ctt gtc cca ttt 290 Met Leu Thr Ser Pro Glu Pro Lys Gly Leu Val Pro Phe 1 5 10 aca aca gag tca ctt gaa ctt ata gaa aat cac att gct aaa aaa tgc 338 Thr Thr Glu Ser Leu Glu Leu Ile Glu Asn His Ile Ala Lys Lys Cys 15 20 25 aat gaa gac ccc gaa gaa gaa gaa ggt tta aaa cca agt cgt aat cta 386 Asn Glu Asp Pro Glu Glu Glu Glu Gly Leu Lys Pro Ser Arg Asn Leu 30 35 40 45 gaa gct ggc aaa aga ctt cca att ccc tat gga acc ctc cct cga gga 434 Glu Ala Gly Lys Arg Leu Pro Ile Pro Tyr Gly Thr Leu Pro Arg Gly 50 55 60 acc gtg tca gag ccc ttg gaa gat gtg gat cca tac tac tat gtt aag 482 Thr Val Ser Glu Pro Leu Glu Asp Val Asp Pro Tyr Tyr Tyr Val Lys 65 70 75 aga aat act ttc atg gtc tta aac aga agc aga gtc atc ttc agg ttc 530 Arg Asn Thr Phe Met Val Leu Asn Arg Ser Arg Val Ile Phe Arg Phe 80 85 90 aat gcg gtt tcc atc ttc tgc aca ttg tct cct cta aac tcc ctc aga 578 Asn Ala Val Ser Ile Phe Cys Thr Leu Ser Pro Leu Asn Ser Leu Arg 95 100 105 aga gca gct atc aag gct ttg gtg cat ccc ctt ttt cgc ctg ctg att 626 Arg Ala Ala Ile Lys Ala Leu Val His Pro Leu Phe Arg Leu Leu Ile 110 115 120 125 tta atc agc gtt ctc act gac agc ata ctt atg tgc atg agt aat cta 674 Leu Ile Ser Val Leu Thr Asp Ser Ile Leu Met Cys Met Ser Asn Leu 130 135 140 cca gaa tgg ata ttg gca ata gag aat act ttg ctt ggg att tac gca 722 Pro Glu Trp Ile Leu Ala Ile Glu Asn Thr Leu Leu Gly Ile Tyr Ala 145 150 155 ttt gaa ata ctt gta aaa gtc att gca aga ggt atc tgg gca ggt tca 770 Phe Glu Ile Leu Val Lys Val Ile Ala Arg Gly Ile Trp Ala Gly Ser 160 165 170 ttt tcc ttc ctt ggg gat ctt tgg aac tgg ctt gat ttc agt gta act 818 Phe Ser Phe Leu Gly Asp Leu Trp Asn Trp Leu Asp Phe Ser Val Thr 175 180 185 ttg ttc gag cta atc aca agg ttt tca cct cta agc tcc ttt tta atg 866 Leu Phe Glu Leu Ile Thr Arg Phe Ser Pro Leu Ser Ser Phe Leu Met 190 195 200 205 ctt aaa act atc aga act ttc cga att ttg aag att atc cct ttg aac 914 Leu Lys Thr Ile Arg Thr Phe Arg Ile Leu Lys Ile Ile Pro Leu Asn 210 215 220 cac ggc ctg cag tct att gtg atg aca ctg gcc cag tgt ttg aag aaa 962 His Gly Leu Gln Ser Ile Val Met Thr Leu Ala Gln Cys Leu Lys Lys 225 230 235 cta ttt ggt gcc att gcc cta gct ctg ttt ttt ctg gct gtg ttt tca 1010 Leu Phe Gly Ala Ile Ala Leu Ala Leu Phe Phe Leu Ala Val Phe Ser 240 245 250 cta ctt gga atg ggc ctc ttc atg ggc aac ctg aag cac aaa tgt ctg 1058 Leu Leu Gly Met Gly Leu Phe Met Gly Asn Leu Lys His Lys Cys Leu 255 260 265 cgg tgg cca gaa gaa aat gaa aat gaa acg ctg cac aac aga act gga 1106 Arg Trp Pro Glu Glu Asn Glu Asn Glu Thr Leu His Asn Arg Thr Gly 270 275 280 285 agc ctt aac tat agt cca gaa aga ata aac ttc tac tac atg gaa gga 1154 Ser Leu Asn Tyr Ser Pro Glu Arg Ile Asn Phe Tyr Tyr Met Glu Gly 290 295 300 gcg aaa tat gct ctc ctt tgc ggc aac agg aca gat gct ggc cag tgt 1202 Ala Lys Tyr Ala Leu Leu Cys Gly Asn Arg Thr Asp Ala Gly Gln Cys 305 310 315 ccg gaa ggt tat gtg tgt gta aaa gaa ggc aca aat cct gac aat ggc 1250 Pro Glu Gly Tyr Val Cys Val Lys Glu Gly Thr Asn Pro Asp Asn Gly 320 325 330 ttc aca agt ttt gac aac ttt ggc tgg tcc ctt ctt gct atg ttt cga 1298 Phe Thr Ser Phe Asp Asn Phe Gly Trp Ser Leu Leu Ala Met Phe Arg 335 340 345 ttg atg aca cag gat tac cct gaa tta ctt tat cac cag atc ctt tat 1346 Leu Met Thr Gln Asp Tyr Pro Glu Leu Leu Tyr His Gln Ile Leu Tyr 350 355 360 365 gct tca gga aag gtc tac atg ata ttt ttt gtt atg atc agt ttc tgg 1394 Ala Ser Gly Lys Val Tyr Met Ile Phe Phe Val Met Ile Ser Phe Trp 370 375 380 ttt gcc ttc tat ttg aca agt ttg ttc ttg ggc ata ctc act atg acc 1442 Phe Ala Phe Tyr Leu Thr Ser Leu Phe Leu Gly Ile Leu Thr Met Thr 385 390 395 tat gaa aag gaa aag cag aga gcc tgt gag gaa tct gga ggc ctt gat 1490 Tyr Glu Lys Glu Lys Gln Arg Ala Cys Glu Glu Ser Gly Gly Leu Asp 400 405 410 ccc aaa tgt caa cag aca gtg aaa gaa ctc gac gaa gaa aat gac gca 1538 Pro Lys Cys Gln Gln Thr Val Lys Glu Leu Asp Glu Glu Asn Asp Ala 415 420 425 gct gag atg gaa act aca caa ata gaa atg aag aaa aga tcc cca act 1586 Ala Glu Met Glu Thr Thr Gln Ile Glu Met Lys Lys Arg Ser Pro Thr 430 435 440 445 tct ata aac acc aca ctg gat ata ctg gaa gac act acc ctc gga cac 1634 Ser Ile Asn Thr Thr Leu Asp Ile Leu Glu Asp Thr Thr Leu Gly His 450 455 460 aga gaa gaa cca gaa aca tcc agg aag aaa tgc cca ata tgt tgg cat 1682 Arg Glu Glu Pro Glu Thr Ser Arg Lys Lys Cys Pro Ile Cys Trp His 465 470 475 aag ttt att aaa acc tgc ttc atc tgg aaa tgc tct ccc tgt tgg gta 1730 Lys Phe Ile Lys Thr Cys Phe Ile Trp Lys Cys Ser Pro Cys Trp Val 480 485 490 aaa ctg aac gag ttt gct gat aga gtt ata aca cac cca ttg gct gat 1778 Lys Leu Asn Glu Phe Ala Asp Arg Val Ile Thr His Pro Leu Ala Asp 495 500 505 ctt ttt ctt gtc atc tgc atc gtt tta aac ata tgc ttc ctc gcc cta 1826 Leu Phe Leu Val Ile Cys Ile Val Leu Asn Ile Cys Phe Leu Ala Leu 510 515 520 525 gaa cat ttt cca atg agc gag gag ctc agg tct ctc ctt cac gtt gga 1874 Glu His Phe Pro Met Ser Glu Glu Leu Arg Ser Leu Leu His Val Gly 530 535 540 aat ttg gtt ttt att gga att tac aca ata gaa ctg att ttg aag ata 1922 Asn Leu Val Phe Ile Gly Ile Tyr Thr Ile Glu Leu Ile Leu Lys Ile 545 550 555 atc gct atg cat cca tat ggg tat ttt caa ata agc tgg aat att ttt 1970 Ile Ala Met His Pro Tyr Gly Tyr Phe Gln Ile Ser Trp Asn Ile Phe 560 565 570 gac agt ata ctt gtg gtt ttg gag tta aca gaa att tta cta gca gat 2018 Asp Ser Ile Leu Val Val Leu Glu Leu Thr Glu Ile Leu Leu Ala Asp 575 580 585 gtt gaa gga cta gct gtt tta ata aca gtc cca ttg ata ttt ata aaa 2066 Val Glu Gly Leu Ala Val Leu Ile Thr Val Pro Leu Ile Phe Ile Lys 590 595 600 605 ctg ggg aag tac gga cca cca ttt aag agt ttg atg cgc atc ctt ggt 2114 Leu Gly Lys Tyr Gly Pro Pro Phe Lys Ser Leu Met Arg Ile Leu Gly 610 615 620 agc tca ttg atg gcc ctg aaa gat ttg gtc ctg ttg ctc tgc ata ttc 2162 Ser Ser Leu Met Ala Leu Lys Asp Leu Val Leu Leu Leu Cys Ile Phe 625 630 635 gtt tac ttc tct gct gtg ttc ggc atg aag ctg ttt ggt cga agt tac 2210 Val Tyr Phe Ser Ala Val Phe Gly Met Lys Leu Phe Gly Arg Ser Tyr 640 645 650 aag gat tgt gtc tgc cac ata aag gaa gac tgc caa ccc caa cgc tgg 2258 Lys Asp Cys Val Cys His Ile Lys Glu Asp Cys Gln Pro Gln Arg Trp 655 660 665 cac atg agt gac ttc ctt cat gcc tac atg acc gtg ttc cga atc ctc 2306 His Met Ser Asp Phe Leu His Ala Tyr Met Thr Val Phe Arg Ile Leu 670 675 680 685 tgt gga gag tgg ata gag aca tta tgg gag tgt atg gag gtt gca ggc 2354 Cys Gly Glu Trp Ile Glu Thr Leu Trp Glu Cys Met Glu Val Ala Gly 690 695 700 cag gcc tgg tgt att cct ttt tac atg atg gtc att tta att gga aac 2402 Gln Ala Trp Cys Ile Pro Phe Tyr Met Met Val Ile Leu Ile Gly Asn 705 710 715 tta ttg ata ctt tac ctc ttt gtg aca ttg gtg agc tct ttc agt tac 2450 Leu Leu Ile Leu Tyr Leu Phe Val Thr Leu Val Ser Ser Phe Ser Tyr 720 725 730 tat gat gct aca tcg gaa gtg aac aaa gaa gcc aaa aac ctt cag ctt 2498 Tyr Asp Ala Thr Ser Glu Val Asn Lys Glu Ala Lys Asn Leu Gln Leu 735 740 745 gcc atg gca agg ata aag tcg gga ata aac tcc atg ctt ctt aaa tta 2546 Ala Met Ala Arg Ile Lys Ser Gly Ile Asn Ser Met Leu Leu Lys Leu 750 755 760 765 atg tgc aca gaa aga agt gtt cct aca gaa gca aca gac caa ata tgt 2594 Met Cys Thr Glu Arg Ser Val Pro Thr Glu Ala Thr Asp Gln Ile Cys 770 775 780 gat cca agt gtt aaa gag aat att tct ggc cat act ctt tct gaa ctg 2642 Asp Pro Ser Val Lys Glu Asn Ile Ser Gly His Thr Leu Ser Glu Leu 785 790 795 agc aac acc caa act ttc ctc aga tat aag gac cag agc agc agc act 2690 Ser Asn Thr Gln Thr Phe Leu Arg Tyr Lys Asp Gln Ser Ser Ser Thr 800 805 810 gag aaa act cca gtg act gaa tct gag agt caa tct ctg att gct agt 2738 Glu Lys Thr Pro Val Thr Glu Ser Glu Ser Gln Ser Leu Ile Ala Ser 815 820 825 ccc agt gcc tct gaa act gtg ccg att gct tca gga gaa tct gat ata 2786 Pro Ser Ala Ser Glu Thr Val Pro Ile Ala Ser Gly Glu Ser Asp Ile 830 835 840 845 gaa aat ctg gat aac aag gag act cgg agc aag tct ggg aat gga ggc 2834 Glu Asn Leu Asp Asn Lys Glu Thr Arg Ser Lys Ser Gly Asn Gly Gly 850 855 860 agt aaa gag aaa atg aag cag tct agc tca tct gag tgc agc aca gtt 2882 Ser Lys Glu Lys Met Lys Gln Ser Ser Ser Ser Glu Cys Ser Thr Val 865 870 875 gat atc gct att tct gaa gaa gaa gaa atg gtc tat gaa cat gaa aag 2930 Asp Ile Ala Ile Ser Glu Glu Glu Glu Met Val Tyr Glu His Glu Lys 880 885 890 tca aag ctt cat aaa aat ggt tat gaa cgc aaa tct tca act ggt caa 2978 Ser Lys Leu His Lys Asn Gly Tyr Glu Arg Lys Ser Ser Thr Gly Gln 895 900 905 atc agt aga gaa tct agg aat gga aag att tgg aaa aac atc agg aaa 3026 Ile Ser Arg Glu Ser Arg Asn Gly Lys Ile Trp Lys Asn Ile Arg Lys 910 915 920 925 act tgc tgc aag ata gta gag aac agc tgg ttt gag tgt ttc att ggc 3074 Thr Cys Cys Lys Ile Val Glu Asn Ser Trp Phe Glu Cys Phe Ile Gly 930 935 940 ctg gtc act ctg ctc tgc aca ggc act ctg gct ctt gaa gac ata tat 3122 Leu Val Thr Leu Leu Cys Thr Gly Thr Leu Ala Leu Glu Asp Ile Tyr 945 950 955 att gat cag aga aaa acc act aaa atc tta ctg gaa tat gcg gac atg 3170 Ile Asp Gln Arg Lys Thr Thr Lys Ile Leu Leu Glu Tyr Ala Asp Met 960 965 970 ata ttt gca tac atc ttc att ctg gag atg ctt ctc aag tgg gtg gct 3218 Ile Phe Ala Tyr Ile Phe Ile Leu Glu Met Leu Leu Lys Trp Val Ala 975 980 985 tat ggc ttt aaa gcc ttt ttc tcc aac aac tgg tac aaa ctg gac ttt 3266 Tyr Gly Phe Lys Ala Phe Phe Ser Asn Asn Trp Tyr Lys Leu Asp Phe 990 995 1000 1005 atg gtt gtt atc gtg ttt tgt ctt agc tta ata ggc aaa act cga gaa 3314 Met Val Val Ile Val Phe Cys Leu Ser Leu Ile Gly Lys Thr Arg Glu 1010 1015 1020 gat ctg aac cct ctg aca tca ata aag ttc ctt cga gca cta aga gtt 3362 Asp Leu Asn Pro Leu Thr Ser Ile Lys Phe Leu Arg Ala Leu Arg Val 1025 1030 1035 ctg tcg cag ttt gaa aga atg aag gtg gtc ctg aga gct ttg ata aaa 3410 Leu Ser Gln Phe Glu Arg Met Lys Val Val Leu Arg Ala Leu Ile Lys 1040 1045 1050 aca acc tta ccc act gtg agc gtg ttt cta gtc tgc cta atg atc tgg 3458 Thr Thr Leu Pro Thr Val Ser Val Phe Leu Val Cys Leu Met Ile Trp 1055 1060 1065 ctg ctt ttc agt gtt att gga gtg cag tta ttt gct ggc aag ttc tat 3506 Leu Leu Phe Ser Val Ile Gly Val Gln Leu Phe Ala Gly Lys Phe Tyr 1070 1075 1080 1085 gaa tgc att gac cca aca aag gga gaa aga ttc cct gta ttt gaa gtt 3554 Glu Cys Ile Asp Pro Thr Lys Gly Glu Arg Phe Pro Val Phe Glu Val 1090 1095 1100 atg aat aaa agt cag tgt gaa aaa ctg tta ttc aat gaa tca atg ccg 3602 Met Asn Lys Ser Gln Cys Glu Lys Leu Leu Phe Asn Glu Ser Met Pro 1105 1110 1115 tgg gag aat gca aaa ctg aac ttt gat aat gtt gga aat ggt ttt ctt 3650 Trp Glu Asn Ala Lys Leu Asn Phe Asp Asn Val Gly Asn Gly Phe Leu 1120 1125 1130 tct tta ctc caa gtg gca aca ttt aat gga tgg atc agt att atg aat 3698 Ser Leu Leu Gln Val Ala Thr Phe Asn Gly Trp Ile Ser Ile Met Asn 1135 1140 1145 tca gca att gat tct gtt ggt gta aac atg cag ccc agc ttt gag tac 3746 Ser Ala Ile Asp Ser Val Gly Val Asn Met Gln Pro Ser Phe Glu Tyr 1150 1155 1160 1165 aac ctc tac atg tat agt tac ttt atc atc ttt gtt atc ttt gga tta 3794 Asn Leu Tyr Met Tyr Ser Tyr Phe Ile Ile Phe Val Ile Phe Gly Leu 1170 1175 1180 ttt ctt cct ctc tgt atg ctg att ggt gtt att att cgc aat ttc aac 3842 Phe Leu Pro Leu Cys Met Leu Ile Gly Val Ile Ile Arg Asn Phe Asn 1185 1190 1195 aag cag aaa att aag cag gga gga tca aac atc ttt ata aca gta aaa 3890 Lys Gln Lys Ile Lys Gln Gly Gly Ser Asn Ile Phe Ile Thr Val Lys 1200 1205 1210 cag aaa aaa cag tac cgg gcc ctg aag aag ctc ttg tat gca gac gtc 3938 Gln Lys Lys Gln Tyr Arg Ala Leu Lys Lys Leu Leu Tyr Ala Asp Val 1215 1220 1225 cag aaa cca aca ccc cgc ccc aga aac aaa ttc caa ggc ttc ctt ttt 3986 Gln Lys Pro Thr Pro Arg Pro Arg Asn Lys Phe Gln Gly Phe Leu Phe 1230 1235 1240 1245 gac cta gta aca cac cgt gtc ttt aat gtt atc atc ata ctt ctt atc 4034 Asp Leu Val Thr His Arg Val Phe Asn Val Ile Ile Ile Leu Leu Ile 1250 1255 1260 tgt ttc caa gca aca acc att atg ata caa aag gat gag cag agt cca 4082 Cys Phe Gln Ala Thr Thr Ile Met Ile Gln Lys Asp Glu Gln Ser Pro 1265 1270 1275 caa atg gaa act gcc atc ttc tgg atg aac tcc att ttt gtc atg ctg 4130 Gln Met Glu Thr Ala Ile Phe Trp Met Asn Ser Ile Phe Val Met Leu 1280 1285 1290 ttc act ctg gaa tgc ata ctg aag ctc act gcc ttc cgt tgc cac tac 4178 Phe Thr Leu Glu Cys Ile Leu Lys Leu Thr Ala Phe Arg Cys His Tyr 1295 1300 1305 ttc acc agt gca tgg aat gtt cat gac ttt atg gtg gtc att ttc tcc 4226 Phe Thr Ser Ala Trp Asn Val His Asp Phe Met Val Val Ile Phe Ser 1310 1315 1320 1325 att aca ggg ctg ctg cta ccc ttg aca ata gga caa tac ttt gtg cct 4274 Ile Thr Gly Leu Leu Leu Pro Leu Thr Ile Gly Gln Tyr Phe Val Pro 1330 1335 1340 cct tcc ctg gtg cag ctg att ctt ctc tct cga gtc atc cac atc ctg 4322 Pro Ser Leu Val Gln Leu Ile Leu Leu Ser Arg Val Ile His Ile Leu 1345 1350 1355 cgt cct ggg aaa gga ccg aag gtg ttc cat gac ctg atg ctt ccc ttg 4370 Arg Pro Gly Lys Gly Pro Lys Val Phe His Asp Leu Met Leu Pro Leu 1360 1365 1370 att ctg gcg ctc cca gca ttg ctg aac att agt ctt ctc atc ttc ctg 4418 Ile Leu Ala Leu Pro Ala Leu Leu Asn Ile Ser Leu Leu Ile Phe Leu 1375 1380 1385 gtc atg ttc atc tac gcc atc ttt gga atg tac aac ttt gcc tat gta 4466 Val Met Phe Ile Tyr Ala Ile Phe Gly Met Tyr Asn Phe Ala Tyr Val 1390 1395 1400 1405 aag aaa gaa gcc gga att aat gat gtg tcc aac ttt gag acc ttt gga 4514 Lys Lys Glu Ala Gly Ile Asn Asp Val Ser Asn Phe Glu Thr Phe Gly 1410 1415 1420 agc agt atg ctc tgt ctc ttc caa gtt aca acg ttt tct ggt tgg gac 4562 Ser Ser Met Leu Cys Leu Phe Gln Val Thr Thr Phe Ser Gly Trp Asp 1425 1430 1435 ggg atg ctg gat gca att ttc aac agt cag tgg tct gac tgc gat cct 4610 Gly Met Leu Asp Ala Ile Phe Asn Ser Gln Trp Ser Asp Cys Asp Pro 1440 1445 1450 gat aaa att aat cca ggg act cag gtc aag gga gat tgt ggg agc cct 4658 Asp Lys Ile Asn Pro Gly Thr Gln Val Lys Gly Asp Cys Gly Ser Pro 1455 1460 1465 tct gtt ggg att tct tat ttt gtc agt tac atc ctc ata tca tgg ttg 4706 Ser Val Gly Ile Ser Tyr Phe Val Ser Tyr Ile Leu Ile Ser Trp Leu 1470 1475 1480 1485 atc att gtt aac atg tac att gtg ttg atc atg gag ttc tta agt att 4754 Ile Ile Val Asn Met Tyr Ile Val Leu Ile Met Glu Phe Leu Ser Ile 1490 1495 1500 cct tct cag aaa aaa agc agg acc ttg agt gaa gat gac ttt agg aga 4802 Pro Ser Gln Lys Lys Ser Arg Thr Leu Ser Glu Asp Asp Phe Arg Arg 1505 1510 1515 ttc ttc cgg gtg tgg aac agg ttt gac cct gat agg acc cag tac ata 4850 Phe Phe Arg Val Trp Asn Arg Phe Asp Pro Asp Arg Thr Gln Tyr Ile 1520 1525 1530 gac tct agc aag ctt tct gat ttt gca gct gct ctg gat cct cct ctt 4898 Asp Ser Ser Lys Leu Ser Asp Phe Ala Ala Ala Leu Asp Pro Pro Leu 1535 1540 1545 ttc atg gca aaa cca aac aag ggc cag ctt gtg gcc atg gat ctc ccc 4946 Phe Met Ala Lys Pro Asn Lys Gly Gln Leu Val Ala Met Asp Leu Pro 1550 1555 1560 1565 atg gct gcg gga gac aga atc cac tgc ctc gac att tta ctt gcc ttt 4994 Met Ala Ala Gly Asp Arg Ile His Cys Leu Asp Ile Leu Leu Ala Phe 1570 1575 1580 acg aaa aga gtg atg gga aag gat gag agg gtg gag aaa atc ctt tca 5042 Thr Lys Arg Val Met Gly Lys Asp Glu Arg Val Glu Lys Ile Leu Ser 1585 1590 1595 gag ata gaa tcc ggg ttc atg tta gcg aac cct ttc aaa atc act tat 5090 Glu Ile Glu Ser Gly Phe Met Leu Ala Asn Pro Phe Lys Ile Thr Tyr 1600 1605 1610 gag ccg att aca act act ttg aaa cgc aaa caa gag gca gtt tca gca 5138 Glu Pro Ile Thr Thr Thr Leu Lys Arg Lys Gln Glu Ala Val Ser Ala 1615 1620 1625 acc atc atc cag cgt gca tat aaa agc tac cgc tta agg caa aat gac 5186 Thr Ile Ile Gln Arg Ala Tyr Lys Ser Tyr Arg Leu Arg Gln Asn Asp 1630 1635 1640 1645 aag aat gta tca gat act cct gct ata gat gac cgc aga gat gat ctt 5234 Lys Asn Val Ser Asp Thr Pro Ala Ile Asp Asp Arg Arg Asp Asp Leu 1650 1655 1660 act tct aaa ggt gct cac tct ggc aaa atc gag gag aag gca tct att 5282 Thr Ser Lys Gly Ala His Ser Gly Lys Ile Glu Glu Lys Ala Ser Ile 1665 1670 1675 cag acc cag att taa tgacacttcc cacttctact ttctttacat atgtccccaa 5337 Gln Thr Gln Ile 1680 gcactaaatg ttaactgatc ttaagctgga gatcagaaac tagagataat gataacatct 5397 gtgtgcccag acatctccat gacaagctca gctttagggt cagtcttctg atgcatcaga 5457 aagacagcag ctcagcgttg ctgcg 5482 3 1681 PRT Mus musculus 3 Met Leu Thr Ser Pro Glu Pro Lys Gly Leu Val Pro Phe Thr Thr Glu 1 5 10 15 Ser Leu Glu Leu Ile Glu Asn His Ile Ala Lys Lys Cys Asn Glu Asp 20 25 30 Pro Glu Glu Glu Glu Gly Leu Lys Pro Ser Arg Asn Leu Glu Ala Gly 35 40 45 Lys Arg Leu Pro Ile Pro Tyr Gly Thr Leu Pro Arg Gly Thr Val Ser 50 55 60 Glu Pro Leu Glu Asp Val Asp Pro Tyr Tyr Tyr Val Lys Arg Asn Thr 65 70 75 80 Phe Met Val Leu Asn Arg Ser Arg Val Ile Phe Arg Phe Asn Ala Val 85 90 95 Ser Ile Phe Cys Thr Leu Ser Pro Leu Asn Ser Leu Arg Arg Ala Ala 100 105 110 Ile Lys Ala Leu Val His Pro Leu Phe Arg Leu Leu Ile Leu Ile Ser 115 120 125 Val Leu Thr Asp Ser Ile Leu Met Cys Met Ser Asn Leu Pro Glu Trp 130 135 140 Ile Leu Ala Ile Glu Asn Thr Leu Leu Gly Ile Tyr Ala Phe Glu Ile 145 150 155 160 Leu Val Lys Val Ile Ala Arg Gly Ile Trp Ala Gly Ser Phe Ser Phe 165 170 175 Leu Gly Asp Leu Trp Asn Trp Leu Asp Phe Ser Val Thr Leu Phe Glu 180 185 190 Leu Ile Thr Arg Phe Ser Pro Leu Ser Ser Phe Leu Met Leu Lys Thr 195 200 205 Ile Arg Thr Phe Arg Ile Leu Lys Ile Ile Pro Leu Asn His Gly Leu 210 215 220 Gln Ser Ile Val Met Thr Leu Ala Gln Cys Leu Lys Lys Leu Phe Gly 225 230 235 240 Ala Ile Ala Leu Ala Leu Phe Phe Leu Ala Val Phe Ser Leu Leu Gly 245 250 255 Met Gly Leu Phe Met Gly Asn Leu Lys His Lys Cys Leu Arg Trp Pro 260 265 270 Glu Glu Asn Glu Asn Glu Thr Leu His Asn Arg Thr Gly Ser Leu Asn 275 280 285 Tyr Ser Pro Glu Arg Ile Asn Phe Tyr Tyr Met Glu Gly Ala Lys Tyr 290 295 300 Ala Leu Leu Cys Gly Asn Arg Thr Asp Ala Gly Gln Cys Pro Glu Gly 305 310 315 320 Tyr Val Cys Val Lys Glu Gly Thr Asn Pro Asp Asn Gly Phe Thr Ser 325 330 335 Phe Asp Asn Phe Gly Trp Ser Leu Leu Ala Met Phe Arg Leu Met Thr 340 345 350 Gln Asp Tyr Pro Glu Leu Leu Tyr His Gln Ile Leu Tyr Ala Ser Gly 355 360 365 Lys Val Tyr Met Ile Phe Phe Val Met Ile Ser Phe Trp Phe Ala Phe 370 375 380 Tyr Leu Thr Ser Leu Phe Leu Gly Ile Leu Thr Met Thr Tyr Glu Lys 385 390 395 400 Glu Lys Gln Arg Ala Cys Glu Glu Ser Gly Gly Leu Asp Pro Lys Cys 405 410 415 Gln Gln Thr Val Lys Glu Leu Asp Glu Glu Asn Asp Ala Ala Glu Met 420 425 430 Glu Thr Thr Gln Ile Glu Met Lys Lys Arg Ser Pro Thr Ser Ile Asn 435 440 445 Thr Thr Leu Asp Ile Leu Glu Asp Thr Thr Leu Gly His Arg Glu Glu 450 455 460 Pro Glu Thr Ser Arg Lys Lys Cys Pro Ile Cys Trp His Lys Phe Ile 465 470 475 480 Lys Thr Cys Phe Ile Trp Lys Cys Ser Pro Cys Trp Val Lys Leu Asn 485 490 495 Glu Phe Ala Asp Arg Val Ile Thr His Pro Leu Ala Asp Leu Phe Leu 500 505 510 Val Ile Cys Ile Val Leu Asn Ile Cys Phe Leu Ala Leu Glu His Phe 515 520 525 Pro Met Ser Glu Glu Leu Arg Ser Leu Leu His Val Gly Asn Leu Val 530 535 540 Phe Ile Gly Ile Tyr Thr Ile Glu Leu Ile Leu Lys Ile Ile Ala Met 545 550 555 560 His Pro Tyr Gly Tyr Phe Gln Ile Ser Trp Asn Ile Phe Asp Ser Ile 565 570 575 Leu Val Val Leu Glu Leu Thr Glu Ile Leu Leu Ala Asp Val Glu Gly 580 585 590 Leu Ala Val Leu Ile Thr Val Pro Leu Ile Phe Ile Lys Leu Gly Lys 595 600 605 Tyr Gly Pro Pro Phe Lys Ser Leu Met Arg Ile Leu Gly Ser Ser Leu 610 615 620 Met Ala Leu Lys Asp Leu Val Leu Leu Leu Cys Ile Phe Val Tyr Phe 625 630 635 640 Ser Ala Val Phe Gly Met Lys Leu Phe Gly Arg Ser Tyr Lys Asp Cys 645 650 655 Val Cys His Ile Lys Glu Asp Cys Gln Pro Gln Arg Trp His Met Ser 660 665 670 Asp Phe Leu His Ala Tyr Met Thr Val Phe Arg Ile Leu Cys Gly Glu 675 680 685 Trp Ile Glu Thr Leu Trp Glu Cys Met Glu Val Ala Gly Gln Ala Trp 690 695 700 Cys Ile Pro Phe Tyr Met Met Val Ile Leu Ile Gly Asn Leu Leu Ile 705 710 715 720 Leu Tyr Leu Phe Val Thr Leu Val Ser Ser Phe Ser Tyr Tyr Asp Ala 725 730 735 Thr Ser Glu Val Asn Lys Glu Ala Lys Asn Leu Gln Leu Ala Met Ala 740 745 750 Arg Ile Lys Ser Gly Ile Asn Ser Met Leu Leu Lys Leu Met Cys Thr 755 760 765 Glu Arg Ser Val Pro Thr Glu Ala Thr Asp Gln Ile Cys Asp Pro Ser 770 775 780 Val Lys Glu Asn Ile Ser Gly His Thr Leu Ser Glu Leu Ser Asn Thr 785 790 795 800 Gln Thr Phe Leu Arg Tyr Lys Asp Gln Ser Ser Ser Thr Glu Lys Thr 805 810 815 Pro Val Thr Glu Ser Glu Ser Gln Ser Leu Ile Ala Ser Pro Ser Ala 820 825 830 Ser Glu Thr Val Pro Ile Ala Ser Gly Glu Ser Asp Ile Glu Asn Leu 835 840 845 Asp Asn Lys Glu Thr Arg Ser Lys Ser Gly Asn Gly Gly Ser Lys Glu 850 855 860 Lys Met Lys Gln Ser Ser Ser Ser Glu Cys Ser Thr Val Asp Ile Ala 865 870 875 880 Ile Ser Glu Glu Glu Glu Met Val Tyr Glu His Glu Lys Ser Lys Leu 885 890 895 His Lys Asn Gly Tyr Glu Arg Lys Ser Ser Thr Gly Gln Ile Ser Arg 900 905 910 Glu Ser Arg Asn Gly Lys Ile Trp Lys Asn Ile Arg Lys Thr Cys Cys 915 920 925 Lys Ile Val Glu Asn Ser Trp Phe Glu Cys Phe Ile Gly Leu Val Thr 930 935 940 Leu Leu Cys Thr Gly Thr Leu Ala Leu Glu Asp Ile Tyr Ile Asp Gln 945 950 955 960 Arg Lys Thr Thr Lys Ile Leu Leu Glu Tyr Ala Asp Met Ile Phe Ala 965 970 975 Tyr Ile Phe Ile Leu Glu Met Leu Leu Lys Trp Val Ala Tyr Gly Phe 980 985 990 Lys Ala Phe Phe Ser Asn Asn Trp Tyr Lys Leu Asp Phe Met Val Val 995 1000 1005 Ile Val Phe Cys Leu Ser Leu Ile Gly Lys Thr Arg Glu Asp Leu Asn 1010 1015 1020 Pro Leu Thr Ser Ile Lys Phe Leu Arg Ala Leu Arg Val Leu Ser Gln 1025 1030 1035 1040 Phe Glu Arg Met Lys Val Val Leu Arg Ala Leu Ile Lys Thr Thr Leu 1045 1050 1055 Pro Thr Val Ser Val Phe Leu Val Cys Leu Met Ile Trp Leu Leu Phe 1060 1065 1070 Ser Val Ile Gly Val Gln Leu Phe Ala Gly Lys Phe Tyr Glu Cys Ile 1075 1080 1085 Asp Pro Thr Lys Gly Glu Arg Phe Pro Val Phe Glu Val Met Asn Lys 1090 1095 1100 Ser Gln Cys Glu Lys Leu Leu Phe Asn Glu Ser Met Pro Trp Glu Asn 1105 1110 1115 1120 Ala Lys Leu Asn Phe Asp Asn Val Gly Asn Gly Phe Leu Ser Leu Leu 1125 1130 1135 Gln Val Ala Thr Phe Asn Gly Trp Ile Ser Ile Met Asn Ser Ala Ile 1140 1145 1150 Asp Ser Val Gly Val Asn Met Gln Pro Ser Phe Glu Tyr Asn Leu Tyr 1155 1160 1165 Met Tyr Ser Tyr Phe Ile Ile Phe Val Ile Phe Gly Leu Phe Leu Pro 1170 1175 1180 Leu Cys Met Leu Ile Gly Val Ile Ile Arg Asn Phe Asn Lys Gln Lys 1185 1190 1195 1200 Ile Lys Gln Gly Gly Ser Asn Ile Phe Ile Thr Val Lys Gln Lys Lys 1205 1210 1215 Gln Tyr Arg Ala Leu Lys Lys Leu Leu Tyr Ala Asp Val Gln Lys Pro 1220 1225 1230 Thr Pro Arg Pro Arg Asn Lys Phe Gln Gly Phe Leu Phe Asp Leu Val 1235 1240 1245 Thr His Arg Val Phe Asn Val Ile Ile Ile Leu Leu Ile Cys Phe Gln 1250 1255 1260 Ala Thr Thr Ile Met Ile Gln Lys Asp Glu Gln Ser Pro Gln Met Glu 1265 1270 1275 1280 Thr Ala Ile Phe Trp Met Asn Ser Ile Phe Val Met Leu Phe Thr Leu 1285 1290 1295 Glu Cys Ile Leu Lys Leu Thr Ala Phe Arg Cys His Tyr Phe Thr Ser 1300 1305 1310 Ala Trp Asn Val His Asp Phe Met Val Val Ile Phe Ser Ile Thr Gly 1315 1320 1325 Leu Leu Leu Pro Leu Thr Ile Gly Gln Tyr Phe Val Pro Pro Ser Leu 1330 1335 1340 Val Gln Leu Ile Leu Leu Ser Arg Val Ile His Ile Leu Arg Pro Gly 1345 1350 1355 1360 Lys Gly Pro Lys Val Phe His Asp Leu Met Leu Pro Leu Ile Leu Ala 1365 1370 1375 Leu Pro Ala Leu Leu Asn Ile Ser Leu Leu Ile Phe Leu Val Met Phe 1380 1385 1390 Ile Tyr Ala Ile Phe Gly Met Tyr Asn Phe Ala Tyr Val Lys Lys Glu 1395 1400 1405 Ala Gly Ile Asn Asp Val Ser Asn Phe Glu Thr Phe Gly Ser Ser Met 1410 1415 1420 Leu Cys Leu Phe Gln Val Thr Thr Phe Ser Gly Trp Asp Gly Met Leu 1425 1430 1435 1440 Asp Ala Ile Phe Asn Ser Gln Trp Ser Asp Cys Asp Pro Asp Lys Ile 1445 1450 1455 Asn Pro Gly Thr Gln Val Lys Gly Asp Cys Gly Ser Pro Ser Val Gly 1460 1465 1470 Ile Ser Tyr Phe Val Ser Tyr Ile Leu Ile Ser Trp Leu Ile Ile Val 1475 1480 1485 Asn Met Tyr Ile Val Leu Ile Met Glu Phe Leu Ser Ile Pro Ser Gln 1490 1495 1500 Lys Lys Ser Arg Thr Leu Ser Glu Asp Asp Phe Arg Arg Phe Phe Arg 1505 1510 1515 1520 Val Trp Asn Arg Phe Asp Pro Asp Arg Thr Gln Tyr Ile Asp Ser Ser 1525 1530 1535 Lys Leu Ser Asp Phe Ala Ala Ala Leu Asp Pro Pro Leu Phe Met Ala 1540 1545 1550 Lys Pro Asn Lys Gly Gln Leu Val Ala Met Asp Leu Pro Met Ala Ala 1555 1560 1565 Gly Asp Arg Ile His Cys Leu Asp Ile Leu Leu Ala Phe Thr Lys Arg 1570 1575 1580 Val Met Gly Lys Asp Glu Arg Val Glu Lys Ile Leu Ser Glu Ile Glu 1585 1590 1595 1600 Ser Gly Phe Met Leu Ala Asn Pro Phe Lys Ile Thr Tyr Glu Pro Ile 1605 1610 1615 Thr Thr Thr Leu Lys Arg Lys Gln Glu Ala Val Ser Ala Thr Ile Ile 1620 1625 1630 Gln Arg Ala Tyr Lys Ser Tyr Arg Leu Arg Gln Asn Asp Lys Asn Val 1635 1640 1645 Ser Asp Thr Pro Ala Ile Asp Asp Arg Arg Asp Asp Leu Thr Ser Lys 1650 1655 1660 Gly Ala His Ser Gly Lys Ile Glu Glu Lys Ala Ser Ile Gln Thr Gln 1665 1670 1675 1680 Ile 4 6927 DNA Mus musculus 4 aagctttact ctcacagaga aaagtcttct gagtgatcaa ttgccaacga tacaacctca 60 ccttagttta ccctgacctg tgaaagatgg ccttcaacag tggagaataa ggagttctag 120 ctgagatgtt tcattaagcg acatattcat ggatcagctt ttgatggcag attttcaggc 180 tccttttctc cactgccaat aattttacaa aacacaattt taaaattgta gtctttatgg 240 gaaacaattc atcctataga tgttgtcaag gacaaaacat tttcactcct gggcagtttt 300 gttgttccct tccctcactg tcatggcgct aaagcggtag ctcatcccca gattaggggc 360 ccaggtgtaa ttgttcttaa gtctgaaatt gtaggggaga gttctttgaa ctcatctctt 420 catgagttca aagaacatat gacaacttat tgatagaatg actttacaca tggccatatt 480 ttacacattt actattttac aggtataaaa ccgaaaatgt tgacttcccc agagccgaag 540 ggccttgtcc catttacaac agagtcactt gaacttatag aaaatcacat tgctaaaaaa 600 tgcaatgaag accccgaaga agaagaaggt ttaaaaccaa gtcgtaatct agaagctggc 660 aaaagacttc caattcccta tggaaccctc cctcgaggaa ccgtgtcaga gcccttggaa 720 gatgtggatc catactacta tgttaagaga aatgtaagta ttaactgtta tcattgaagc 780 tatattttac ttcgcttata ttcagccact tgaaatgtaa ttgagataag acttaaagaa 840 aattaataga gaaggcattc tttcataatc tattctttgt gggggtcaac atgctcaaga 900 tagttaaacc tgataaaata tctgagtaat atattatggt taatgaccgt agtatatata 960 ctgctattcc ttaatataag tggctattgt gaaaatatgc taattaccat tttctgatta 1020 gcaattttaa aacaatcatg aaatatttag aatatggaca gaaatttcaa ataccttgat 1080 aacttactag tcaaaacagt acatttattt ttaatcatat ataaatccac aaattcaaac 1140 ctccctcatt tccaggaaga ctttagagac ctagaaatta tgtatacaca aacacacaca 1200 cacacataca cacacacaca cgcacacgca aatgcacacc ctaccatcac aaacacaaaa 1260 taagtacaag aatgatttct gttagaaaat tcagacatgt ggattgatga agatagatga 1320 gtcttgtttc aaaagcatgg tttgggggct ggagaaatgt ctcagtttct aatagcactg 1380 gctgttcttc tggacgtcat aagattgatt gcagtccctg tacagtaggt cacagccttt 1440 tgtaactcta gttccagaag atttgacctt cttttggcca ctatgggcac tgggaatgca 1500 aataattcat atatgcacac agtgaagaca tctatatact tgaaatgaga taaaatttta 1560 ggtacagctt gcagaatact tggaatttta ataaagccaa ggtagaacag tttaaggaca 1620 aatggaatgg caacagccaa gattgattct acaagagggc atagaaaggg ctgtgctatt 1680 actggaaaat cagttatgtt gttgactgtg gcaaacatga gagagtggag gtgtcatcat 1740 ggaattactg tagtggacaa ggtcatttgg ggtgaatgtg gcagatgaat aaaaccaagc 1800 taataccttt cttaattaaa agaaaagtga tcaatacaga aaataaaata gaacataaaa 1860 gacagggaga aatataaggt agagaagagg aggagagaaa gagatgaggg agaagagaag 1920 gggtgagaat gggaagagga agagaagaga gataggtaga aatgaagaga ggggaggaag 1980 ggacttgtat attacagtta atttacctag tgtacaaact gatagttctt agtataaatg 2040 gcttttatgt aaatatgatg attactgtct tctaattatc tatttttaaa taatcataaa 2100 atatttacac tataaaagaa aaagagagaa ggaggagaga aagaacaaga aggaaatagg 2160 gtagagggaa ccctgagatt cagtgtctat gtcaggaaaa gaaacagtaa aatatgactg 2220 atgaaaaatg ccaatgtctg tagcttgaaa gagtgaagtt acattgacag aaagtgaaat 2280 aaagtttatt tactagcatc ttgatattca tgtatcatat tcactgatgt tatatctaat 2340 gaggagatga gactgaacga aaatatctgc agaaaaatac attcattgca taactgttcc 2400 tataataatg tcatggtgtc atttgaaata ttttaaggac attttagtta aaatgcaagt 2460 tcagtcctca tttgtattgt tctggcactc actttgtggt gtgcttgagt tgataatggc 2520 cagtaacctt aggattgcct gtcaatattg cagccatctt aatactgagt aaggtatgca 2580 ggcatgctag acatgggaat actgccattg aagataaaat caaagctctt aagaaacaaa 2640 acaaatactt tatgacaggc cccatgtcca gcagtagttg gccaaaaaaa aaaaaaaaaa 2700 ttgtgatttt gattcttttg tcagggtggc atgtttggag acagggattc cctgagtagc 2760 cctggctgtc ctggaactca ctctgcagtc caggctggcc tcgaactcag agatctgctt 2820 gcttctgtct tctgagttct aggattacgg gtataagttt ttattaaaag tataaggctt 2880 tgctttttgt tgttgttttt ttttgtttgt ttgtttgttt tgttttttgg ttggttgttt 2940 ttgttgttgt tggtggtggt ggtggtagtg gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 3000 gtgtgtgtgt gtgtgtgtgt gtgtggaggg agagagagag tgtgctgttt ctgttggaat 3060 tttttgtcat ttttctttaa cttgttatgt tttggtttct ttgagaaaga atgaaggaac 3120 ctgaagttgg ttggatacgg gtgtggggag gatcttggaa tgagttgaga aaggacaaag 3180 aatataatca aagcttacta tatgaaaaat taaataaaat gtgaaataac tacaaaaatc 3240 tcaaaacaac attttgagtg atttaggatc ctagcagcta tgaggcccat gggttgaaat 3300 ttgccagtga cttcatgcac tctaaagcct gcaaatgtga cttcatgaac tctgaagcct 3360 gccagggtga cctcatgctc tttgaagcct gactgggtga ctttatgcac tcagtgctgc 3420 tctcagtaac atcacactgt tcaagtatct gggttgtatt tggcttactc tcttacacat 3480 agcacagaga caaataatca taaaattaag actatgtatg aaatcaggag aaaacctgaa 3540 agtacgcatc ccttcacaat gacacacaca tatattgaac tccacatgga gtctgcaatt 3600 gttttggaag ctactatgga aagtagaatt tacacaacca ggtatattat ttgtttctac 3660 ctgttttatt taagtggaga tgggagaata ggtacaatag ttaaacaaag cctcatagca 3720 gttatatatt gaactacact tatctgagaa ttgagaccca gagtagattc cagttagagc 3780 agacatgcat ggagaagtgt tggcagttgt atattcttat aatttctttt tcatatgcgt 3840 ctgtcaacat aggaagcaat aagtcatacc tcctatatca ttatcagaat atatggtaga 3900 ctgtatagtc tattctattg catataagat taatttttaa gccaggcctt ctggcagggc 3960 tataattctc aaacaacaaa gtattgcaga ttcaagtcct ctgtggtcta aactgtgagt 4020 tcaaggtcag ccttaacaat ttaatgaaat ctattttcaa gagaaaaaga atataaagat 4080 cctcaggaga aataatggaa gagagtttgc ccagcacaca tgaggcccct gtcacagagg 4140 aagaaaatat tacatatttt ttatttataa aaatatagtt atcttattat ataacatgct 4200 atgttatgtt atgttttatg ttataacaca tgacatttaa tctcatagtt ttcagaatta 4260 actggatttt cagtgttagc tggatcaaac tcagggcctg ctgaagggtg aacaaacact 4320 gttcttcaag ttacattacc agacagtaca tgttaatgca cccctacatt aatactttct 4380 ctgaagggac atatatagta aacacacaca cacacacaca cacacatata tatatatata 4440 catatacaca cacacatata tatatacact ttcaattacc atatataata tataccatat 4500 acattatagt aatgttcatt tatatgaaca aatgtaaaaa tgagcacaca tatgtatgaa 4560 tgtacataag cccacatgta catatgtata aataagccca tccacctacc tatatattga 4620 acaatgtctg ttttagaata tttatgtttc ttactatatt tattaagact tagtacttta 4680 gtatccacaa tccttgtgtt tatattacat aaattactat ttttagatag gttcatatca 4740 ataatattaa ataagttagg ttttgtaaaa acattaattt ttaggattta ccttgtaatt 4800 gttttatttt tttattcgat attttcttta tttacatttc aaatgctttc ccgaaagtcc 4860 cctataccct cccacctccc tgctccccta ctcacccact cccatttctt ggccctggtg 4920 ttcccctgta ttggggcata taaagtttgc aagaccaagg ggtctctatt cccaatgatg 4980 gccgactagg ccatcttctg ctacatattc agctagagac acgagctctg gggatactgg 5040 ttaataattg ttttattttt aatatatttt atttaaaata gaattacata aattatcctc 5100 ttattttctc ccctacagtg tctccctcat aaataatata aacaattgat gtgagatttg 5160 accattgctt ttataacact tcattgactt tttttctgat ataaaattaa aatttttaca 5220 ttcattaagt tatgagataa aggccctctg atgctttgaa tgcaaatcca cacaccatct 5280 gaagagtttc tttttcatta gttcaaggtg tgattgcacc ataatgactt tcttaagtac 5340 aaaccagcaa aaaataaata aattaattaa ttattaaaat aaaataagtg gtgagctatc 5400 aggcaagtcc aaatataagt gaaaactact ccctttgctt taaaaacaaa tgtcaaagcc 5460 aatgaaaata tgaaataatt tcaaactggt gagcattggt aatatgttgc ttcagttctc 5520 cattctgttt gttaatgtct ttcctgtggt tccttacaga ctttcatggt cttaaacaga 5580 agcagagtca tcttcaggtt caatgcggtt tccatcttct gcacattgtc tcctctaaac 5640 tccctcagaa gagcagctat caaggctttg gtgcatccat atcctttcaa agtgtgatgg 5700 gttgtgtcat cggcatacta aaaataagtc ctgatgttct gtcattcaac ccctgttgta 5760 gtaatattta acaaatatca attcttattc ttcaaataga atgacatgta ttttcaatat 5820 ttacaaaaga atctcgcccc ttatatctac agatgcaatt tactggtctt ttgtaatgtg 5880 atttcttctc cattattcct tgaccctggc ttaccctttt tcgcctgctg attttaatca 5940 gcgttctcac tgacagcata cttatgtgca tgagtaatct accagaatgg atattggcaa 6000 tagagtaagt tacttagttt tgctatatat aataaagtct gtaattatat tttggtttta 6060 acattttaat atttattgta ttccattaaa ttacaaaaac atgttatgaa atgaaacatg 6120 atactattta aatttttaaa tgtttaaaaa agttataaag acaagaggtt tgtttcactc 6180 acagctttga aggttcaaga gcacctatgc tgttcagctt cagggacaat taggtcctct 6240 cacctaatta acctgtggcc tgtggaaaca ggagaagctc tttcagaagc agccattcat 6300 gtcttgatgc tgtaagccca tgactggtat gaagcctgcc acaacttttc tcttactctt 6360 cttgtgggtc ttgtgcaaac aagcaggcat gcgctccagt gccacaagga ccttcaagtg 6420 caccccacgt ctagtgctcc gtaaactctt gcctcaccat gatcgggatc aagttctaaa 6480 tatgtagaat attgtagaca tctgaaaaca aacctcatct tcatatttct tttctgtata 6540 tactcgtatg tttgtggggc atagcaatat gaacaccatg actaaatttt ctaaataaaa 6600 caaacatgta tatgcacata cacatatata tagatgtata ttcatcaatt tggggtttta 6660 ttttgccctt tcattcttct tacttaattc ctgttggtgt tattttattt gattcatgtt 6720 ctttatacca cttgcctttg actcctgttc cagactcttg aatcctggaa ttacatcttc 6780 ttatcacgac tttacatatc tacagttcta tatagactga aggtttaatt atattaatta 6840 attatgttca ttggaatatt taggtccttt gctaaatgta tataccatgt ttcacctcat 6900 gcttgttttc ttccttattt aaagctt 6927 5 20 DNA Artificial Sequence Description of Artificial SequencePrimer1 5 atgttgactt ccccagagcc 20 6 20 DNA Artificial Sequence Description of Artificial SequencePrimer2 6 aaccaggcaa agcgccattc 20 7 21 DNA Artificial Sequence Description of Artificial SequencePrimer3 7 catcttccaa gggctctgac a 21 8 16 PRT Rattus norvegicus 8 Ser Val Ser Glu Thr Val Pro Ile Ala Ser Gly Glu Ser Asp Ile Lys 1 5 10 15 

What is claimed is:
 1. A null mutant non-human animal characterized in showing salt intake behavior similar to that of wild-type animals under water-sufficient conditions and showing much more intakes of hypertonic saline compared with wild-type animals under water- and salt-depleted conditions.
 2. The null mutant non-human animal according to claim 1, wherein the function of Na_(v)2 gene is deficient on its chromosome.
 3. The null mutant non-human animal according to claim 2, wherein the non-human animal is a rodent.
 4. The null mutant non-human animal according to claim 3, wherein the rodent is a mouse.
 5. A gene that codes for a protein acting as a sensor of extracellular sodium ion level.
 6. The gene according to claim 5, wherein the protein is comprised of amino acid sequence shown in Seq. ID No. 3, or is comprised of amino acid sequence where one or a few amino acids are deficient, substituted, or added, in amino acid sequence shown in Seq. ID No.
 3. 7. The gene according to claim 5, which is comprised of DNA that contains a base sequence shown in Seq. ID No. 2 or its complimentary sequence, and a part or whole of those sequences.
 8. The gene according to claim 5, which is comprised of DNA being hybridized under stringent conditions with DNA that contains a base sequence shown in Seq. ID No. 2 or its complimentary sequence, and a part of or whole of those sequences.
 9. A protein acting as a sensor of extracellular sodium ion level.
 10. The protein according to claim 9, which is comprised of amino acid sequence shown in Seq. ID No.
 3. 11. The protein according to claim 9, which is comprised of amino acid sequence where one or a few amino acids are deficient, substituted, or added, in amino acid sequence shown in Seq. ID No.
 3. 12. A fusion protein created by combining a protein acting as a sensor of extracellular sodium ion level and a marker protein and/or a peptide tag.
 13. The fusion protein according to claim 12, wherein the protein acting as a sensor of extracellular sodium ion level is the protein according to claims 10 or
 11. 14. An antibody which specifically combines with a protein acting as a sensor of extracellular sodium ion level.
 15. The antibody according to claim 14, wherein the protein acting as a sensor of extracellular sodium ion level is the protein according to claims 10 or
 11. 16. The antibody according to claims 14 or 15, wherein the antibody is a monoclonal antibody.
 17. A host cell which contains an expression system that can express a protein acting as a sensor of extracellular sodium ion level.
 18. The host cell according to claim 17, wherein the protein acting as a sensor of extracellular sodium ion level is the protein according to claims 10 or
 11. 19. A transgenic non-human animal which excessively expresses a protein acting as a sensor of extracellular sodium ion level.
 20. The transgenic non-human animal according to claim 19, wherein the protein acting as a sensor of extracellular sodium ion level is the protein according to claims 10 or
 11. 21. The transgenic non-human animal according to claims 19 or 20, wherein the non-human animal is a mouse or a rat.
 22. A method of screening a material that promotes or suppresses the function or the expression of a protein acting as a sensor of extracellular sodium ion level characterized in using a cell that expresses a protein acting as a sensor of extracellular sodium ion level, and a subject material.
 23. The method of screening a material that promotes or suppresses the function or the expression of a protein acting as a sensor of extracellular sodium ion level according to claim 22, wherein the cell that expresses a protein acting as a sensor of extracellular sodium ion level is the host cell according to claims 17 or
 18. 24. A method of screening a material that promotes or suppresses the function or the expression of a protein acting as a sensor of extracellular sodium ion level characterized in using the non-human animal according to any one of claims 1 to 4 or the non-human animal according to any one of claims 19 to 21, and a subject material.
 25. A material that promotes or suppresses the function or the expression of a protein acting as a sensor of extracellular sodium ion level characterized in being available through the screening method according to any one of claims 22 to
 24. 26. A medical compound used for curing patients who need promotion of the function or enhancement of the expression of a protein acting as a sensor of extracellular sodium ion level, and containing the protein according to any one of claims 9 to 11 or the material that promotes the function or the expression of a protein acting as a sensor of extracellular sodium ion level according to claim 25 as its effective components.
 27. A medical compound used for curing patients who need suppression of the function or the expression of a protein acting as a sensor of extracellular sodium ion level, and containing the protein according to any one of claims 9 to 11 or the material that suppresses the function or the expression of a protein acting as a sensor of extracellular sodium ion level according to claim 25 as its effective components. 